Identical Genomic Organizat

Current Biology 22, 2053–2058, November 6, 2012 ª2012 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2012.08.052

Reportion

of Two Hemichordate Hox Clusters

Robert Freeman,1,12 Tetsuro Ikuta,2,12 Michael Wu,3

Ryo Koyanagi,2 Takeshi Kawashima,2 Kunifumi Tagawa,4

Tom Humphreys,5 Guang-Chen Fang,6 Asao Fujiyama,7

Hidetoshi Saiga,8 Christopher Lowe,9 Kim Worley,10

Jerry Jenkins,11 Jeremy Schmutz,11 Marc Kirschner,1

Daniel Rokhsar,3 Nori Satoh,2,* and John Gerhart3,*1Department of Systems Biology, Harvard Medical School,Boston, MA 02114, USA2Marine Genomics Unit, Okinawa Institute of Science andTechnology Graduate University, Onna,Okinawa 904-0495, Japan3Department of Molecular and Cell Biology, University ofCalifornia, Berkeley, Berkeley, CA 94720-3200, USA4Marine Biological Laboratory, Graduate School of Science,Hiroshima University, Onomichi, Hiroshima 722-0073, Japan5Department of Cell and Molecular Biology, University ofHawaii, HI 96822, USA6Clemson University Genomics Institute, Clemson,SC 29634, USA7National Institute of Genetics, Mishima,Shizuoka 411-8540, Japan8Department of Biological Sciences, Graduate Schools ofScience and Engineering, Tokyo Metropolitan University,Hachiohji, Tokyo 192-0397, Japan9Department of Biology, Stanford University, Hopkins MarineStation, Pacific Grove, CA 93950, USA10Department of Molecular and Human Genetics, HumanGenome Sequencing Center, Baylor College of Medicine,Houston, TX 77030, USA11HudsonAlpha Institute for Biotechnology, 601 GenomeWay,Huntsville, AL 35806, USA

Summary

Genomic comparisons of chordates, hemichordates, and

echinoderms can inform hypotheses for the evolution ofthese strikingly different phyla from the last common

deuterostome ancestor [1–5]. Because hox genes playpivotal developmental roles in bilaterian animals [6–8], we

analyzed the Hox complexes of two hemichordate genomes.We find thatSaccoglossus kowalevskii andPtychodera flava

both possess 12-gene clusters, with mir10 between hox4and hox5, in 550 kb and 452 kb intervals, respectively. Genes

hox1–hox9/10 of the clusters are in the same genomic orderand transcriptional orientation as their orthologs in chor-

dates, with hox1 at the 30 end of the cluster. At the 50 end,each cluster contains three posterior genes specific to

Ambulacraria (the hemichordate-echinoderm clade), twoforming an inverted terminal pair. In contrast, the echino-

derm Strongylocentrotus purpuratus contains a 588 kbcluster [9] of 11 orthologs of the hemichordate genes,

ordered differently, plausibly reflecting rearrangements ofan ancestral hemichordate-like ambulacrarian cluster. Hox

clusters of vertebrates and the basal chordate amphioxus

12These authors contributed equally to this work

*Correspondence: norisky@oist.jp (N.S.), jgerhart@berkeley.edu (J.G.)

[10] have similar organization to the hemichordate cluster,

but with different posterior genes. These results providegenomic evidence for a well-ordered complex in the deutero-

stome ancestor for the hox1–hox9/10 region, with thenumber and kind of posterior genes still to be elucidated.

Results and Discussion

Herewe characterize the order, transcriptional orientation, andclustering of the Hox genes of the genomes of two widelystudied model hemichordates, Saccoglossus kowalevskiiand Ptychodera flava [4, 11, 12], that represent two majorevolutionary branches of enteropneust hemichordates thatdiverged an estimated 400 million years ago (MYa) [13]:Saccoglossus, of the direct developing harimaniids, andPtychodera, of the indirect developing ptychoderids [14].Both are vermiform marine animals with gill slits, a nonseg-mented body, and a muscular proboscis for burrowing (so-called ‘‘acorn worms’’). A third branch, not included here, isthat of pterobranch hemichordates, which may be an offshootof the harimaniids or basal to both branches [14]. Together, thephyla of hemichordates and echinoderms (diverged an esti-mated 540 MYa [13]) comprise the Ambulacraria, sharinga last common ambulacrarian ancestor. Similarly, the Ambula-craria and the chordate phylum comprise the Deuterostomesuperclade, sharing a last common deuterostome ancestor.The similarities of Hox genes and their clusters across thesegroups can, if found, provide information about the Hoxcomplement of the ambulacrarian and deuterostome ances-tors that gave rise to these three diverse phyla [3–5].

Characterization of the Hox Genes of S. kowalevskiiand P. flava

Following the isolation of ANTP-class Hox gene sequencesfrom the sea urchin Strongylocentrotus purpuratus [15],an echinoderm, complementary DNA (cDNA) clones wereobtained from hemichordates for 11 of 12 Hox genes inS. kowalevskii (excepting hox8) [16, 17], and 8 of 12 inP. flava (excepting hox2, hox3, hox7, and hox8) [18]. At thetime, there was no information on the genomic linkage ofHox genes in hemichordates. For hemichordate genes hox1–hox8, numerical identities were preliminarily assigned basedon the best match of the homeodomain sequence to paralo-gous groups of vertebrate Hox genes and to the sea urchingenes. Hox9/10 and hox11/13a,b,c, which were first namedin S. purpuratus [15] because of their broad similarity tovertebrate posterior genes, were used as gene names forsubsequently discovered ambulacrarian orthologs. The hox1–hox11/13c (except for hox8) genes of S. kowalevskii areexpressed along the anteroposterior axis of embryonic ecto-derm and juvenile epidermis [16, 17] in the same order as theirassigned numerical gene names, and similar to the orderedexpression of the orthologous Hox genes in the vertebratecentral nervous system and in the ectoderm of protostomessuch as Drosophila [6, 7].In the course of our genomic analysis, we obtained the

previously uncharacterized Hox genes of S. kowalevskii andP. flava (see Supplemental Experimental Procedures available

Figure 1. Molecular Phylogenetics of the Hemichordate Hox Genes

A maximum-likelihood phylogram was constructed from the 60-amino-acid

homeodomain sequences from64Hoxgenes, and using twoNK2.1 genes as

anoutgroup. TheHoxgenesof the hemichordatesP. flava andS. kowalevskii

are shown in blue. Red circles indicate genes characterized anew in this

study. PhyML was used (www.phylogeny.fr/version2_cgi/one_task.cgi?

task_type=phyml; see Supplemental Experimental Procedures). The aLRT

branch support values are shown as percentage in red. Branches with

support values under 50% were collapsed. Branch length is indicated by

thebar in the lower right. Pf,Ptychoderaflava; Sk,Saccoglossus kowalevskii;

Sp, Strongylocentrotus purpuratus; Mr, Metacrinus rotundus (for hox4,

which is absent in S. purpuratus); Am, Branchiostoma floridae (amphioxus);

and Mm, Mus musculus.

See also Figures S1 and S2 and Table S1.

Current Biology Vol 22 No 212054

online). The total complements of 12 genes each for thesespecies are summarized in the phylogenetic tree of Figure 1and the homeodomain sequence alignments of Figures S1and S2. Membership of each gene in a vertebrate-definedparalogous group was further assessed by submitting thehomeodomain sequences to HoxPred [19]. From theseanalyses, hox1–hox8 of the two hemichordates are found tostrongly resemble each other as well as hox genes of echino-derms and chordates. The hemichordate sets contain: (1)two anterior genes (hox1 and hox2) probably descendedfrom PG1 and PG2 orthologs dating back to the bilaterianancestor, (2) a group3 gene (hox3) probably descended froma PG3 ortholog of that ancestor, and (3) five central genes(hox4, hox5, hox6, hox7, hox8) that can be matched with chor-date counterparts.The four posterior genes (hox9/10, hox11/13a,b,c) of the

hemichordate sets, although clearly similar to each other andto the echinoderm posterior genes and although having PG9character, have only poorly supported phylogenetic relation-ships to specific chordate posterior genes, as others havenoted [15, 20]. From phylogenetic analysis of the 60 aa home-odomains (Figure 1) as well as of 75 aa blocks encompassingthe homeodomain and conserved among posterior hox genes(Figure S3), we find that the hemichordate and echinodermhox9/10 genes, in keeping with their name, group with verte-brate hox9 and somewhat less well with vertebrate hox10,and Amphioxus hox9 and hox10, whereas the other genes(hox11/13a,b,c of ambulacraria) form a phylogenetic groupseparate from those of vertebrate hox11–hox14 and amphi-oxus hox13–15, and appear to have diverged within theambulacrarian lineage (Figures S2 and S3). Lacking evidencefor orthology to vertebrate hox11, hox12, hox13, and withevidence for their close-relatedness to the echinoderm genes,we suggest that the 11/13a,b,c genes be renamed asambPa,b,c (ambulacrarian Posterior a,b,c). The extensivediversification of posterior gene sets in the different lineagesof deuterostomes, as compared to their anterior and centralgene sets, has been attributed to ‘‘deuterostome posteriorflexibility’’ [20] and to multiple independent duplications [19],as discussed later.

Assembly of the Hox Complexes

For both hemichordates, the genomic analysis entailed theisolation of overlapping BAC sequences carrying subsets ofhox genes, complemented by the assembly of whole genomeshotgun sequences to produce large scaffolds or supercon-tigs containing the entire clusters (see Supplemental Experi-mental Procedures). For S. kowalevskii a single scaffold(Scaffold_166 of 951 kb; Figure 2A) contains the 12-genecluster. Nine genes (hox1 to hox9/10) are arranged in thesame order and transcriptional orientation as their vertebrateorthologs, with hox1 at the 30end, and with three posteriorgenes at the 50 end, namely, hox11/13a in tandem with hox1–9/10 but hox11/13c and 11/13b inverted as a terminal pair.The entire cluster, from exon2 of hox1 to exon2 of the inverselyoriented hox11/13b, spans a genomic distance of 550 kb.A hox8-like homeodomain sequence was recovered in theinterval between hox7 and hox9/10 (deposited as a GNOMONmodel [GenBank GI291221533]). Furthermore, a mir10 se-quence was found between hox4 and hox5.For P. flava, four BAC clones and three genome shotgun-

derived contigs were linked to form a supercontig of 455.7 kb,which included the 12 P. flava Hox genes within a 451.7 kb-long genomic region (Figure 2B). Remarkably, the organization

Figure 2. Clustering of Hox Genes in Hemichordate Genomes

Genomic regions w500 kb-long are shown for (A) Saccoglussus kowalevskii and (B) Ptychodera flava. Both contain the 12 genes Hox1 to Hox11/13b (blue

arrows in the direction of transcription). In both, the ten genes Hox1–Hox11/13a are aligned in the same direction, whereas two genes, Hox11/13c and

Hox11/13b, are in the opposite direction. Red bars indicate the position of mir10 genes, and the orange bar in (B) indicates a gap. BAC clones are shown

as green lines, scaffolds or contigs obtained from whole-genome shotgun reads as purple lines, and PCR-amplified fragments as brown lines.

See also Figure S2.

Genomic Clustering of Hemichordate Hox Genes2055

of the cluster is the sameas that ofS. kowalevskii, namely, hox1to hox9/10 are arranged in the same order and transcriptionalorientation as their vertebrate orthologs, with hox1 at the30end, and three posterior genes are arranged at the 50 end,with hox11/13a in tandem with hox1-9/10 but hox11/13c andhox11/13b inverted as a terminal pair. A mir10 sequenceresides between hox4 and hox5.

From the assembled complexes we estimated the exon-intron structure, size, and intergenic distances of the 12 genesof each (TablesS1AandS1B). ForS. kowalevskii, all geneshavetwo exons except hox9/10, which has four, and all homeoboxsequences are contained within single exons. The averageprimary transcript size and messenger RNA (mRNA) size forthe 12 genes are 5,698 and 2,651 nucleotides, respectively(based on cDNA sequences placed against the Scaffold_166sequence; Supplemental Experimental Procedures). An inter-genic distance of 127 kb separates hox11/13a and hox11/13c; that is, it separates the group of ten tandemly orderedgenes (hox1–hox11/13a) from the inverted pair (hox11/13cand hox11/13b). When this interval is omitted, the average in-tergenicdistance is36kb for the remaininggenesof thecluster.

For the P. flava complex, all genes have two exons excepthox5 and hox7, which have three, and all homeobox se-quences are contained in single exons (the estimates ofdifferent numbers of exons for hox5, hox7, and hox9 betweenthe two species are not definitive because they are based oncDNA analysis in some cases and on gene prediction in others;Tables S1A and S1B). The average mRNA size is 1,691 nucle-otides in the P. flava complex. The average intergenic distanceis 34 kb for all genes of the cluster. Like S. kowalevskii, thelargest (66 kb) intergenic space occurs between the group often tandem genes (hox1–hox11/13a) and the inverted pair(hox11/13c and hox11/13b).In both complexes, no other protein-coding gene was iden-

tified within the cluster by routine sequence searches (Figures2A and 2B). However, for S. kowalevskii, two sense and twoantisense noncoding transcripts of unknown function havebeen detected in and near the cluster (Table S2; SupplementalExperimental Procedures). Outside the S. kowalevskii cluster,an astacin-like protease sequence and a thioredoxin4-likesequence reside within 11 kb and 47 kb, respectively, of theterminal hox11/13b gene. However, within the 110 kb interval

Figure 3. Genomic Organization of Two Hemichordate Hox Clusters and Hypothetical Reconstructions of Hox Cluster of Ambulacrarian Ancestor

Summary of Hox clusters of extant two hemichordates and one echinoderm (Ambulacraria) and two hypothetical reconstructions of the cluster of the am-

bulacrarian ancestor, arranged in a phylogenetic tree [1]. Hox gene location and transcriptional orientation are indicated by triangles, the colors of which

represent the different categories of Hox genes: anterior (purple), group3 (yellow), central (green), and posterior (red). The mir10 locus is indicated by

name, between hox4 and hox5. Note the identical organization of the five gene segment (hox6, hox7, hox8, hox9/10, hox11/13a) of the echinoderm

(S. purpuratus, sea urchin) and hemichordate clusters (bounded by dashed lines). In the hypothetical cluster 1 (upper left), the minimal ancestral cluster

is proposed to be the same as the extant hemichordate clusters, including the inverted hox11/13b,c pair of genes, Then, within the lineage to sea urchins,

the cluster underwent rearrangements and gene loss (hox4) as well as an inversion and translocation of hox11/13c. In the hypothetical cluster 2 (lower left),

the minimal ancestral cluster is proposed to be like that of hemichordates except that hox11/13b and hox11/13c are in tandem with the other genes, not

inverted as a pair. Then within the echinoderm lineage, the cluster underwent rearrangements of hox1–5 and gene loss (hox4), as well as an inversion,

but not translocation of hox11/13b, whereas hox11/13b and hox11/13c inverted as a pair in the hemichordate lineage. The proposal of the kinds of hox genes

in the ancestral cluster is supported by the hox sequence repertoire across extant Ambulacraria [6, 18, 20, 21]. Whereas hox1–9 (called 9/10 in Ambulacraria)

are orthologous to chordate genes 1–9, the ambulacrarian posterior genes 11/13a,b,c differ from vertebrate genes 11–13 and are identified here as

AmbPa,b,c to indicate their ambulacrarian-specific ancestry.

See also Figures S3 and S4.

Current Biology Vol 22 No 212056

from hox11/13b to the end of Scaffold_166, no evx sequencewas found, whereas evx is located adjacent to posterior genesof vertebrate clusters A and D, the amphioxus cluster, and thesea urchin cluster. TheS. kowalevskii evx gene is on a separateScaffold_145 (954 kb), 120 kb from one end; we have noevidence linking this scaffold to that of the Hox cluster.

In summary, these two hemichordate species, separatedby an estimated 400 MYa [14], possess 12-gene Hox clustersof identical organization, with nine genes (hox1–hox9/10)arranged in the same genomic order and transcriptional orien-tation as their orthologs in chordates, with hox1 at the 30 endof the cluster. At the 50 end, both clusters contain three poste-rior genes specific to Ambulacraria, two forming an invertedterminal pair. The cluster sizes are 550 and 452 kb, respec-tively, for S. kowalevskii and P. flava.

Comparison of Deuterostome Hox ClustersFigure 3 summarizes the genomic Hox clusters for the twohemichordates reported here and for the sole echinodermexample, the 11-gene cluster of S. purpuratus, which has adifferent order and orientation of genes from the hemichordateclusters. These differences can be interpreted as arising byway of rearrangements of an ancestral ambulacrarian clusterhaving hemichordate-like organization (Figure 3, hypotheticalcluster 1). That is, five genes in the middle of the echinodermcluster (hox6, hox7, hox8, hox9/10, hox11/13a) have thesame order and orientation as their orthologs in the hemichor-date clusters (Figure 3; dotted lines). If within the lineage to seaurchins, the ancestral ambulacrarian cluster underwent aninversion of the (hox1, hox2, hox3, hox4, mir10, hox5) groupof genes at the 30 end of the cluster, and then a translocationof the inverted genes (hox1, hox2, hox3, hox4, mir10) but not

hox5 to the 50 end of the cluster, followed by loss of hox4but retention of mir10, plus an inversion and translocation ofhox11/13c, the outcome would be the present-day echino-derm S. purpuratus cluster of mixed orders and orientations.The loss of hox4 presumably occurred after the echinoidlineage split from crinoids and asteroids, which have hox4[21, 22]. This and other rearrangement schemes have beenpreviously discussed [9, 15].From the comparisons of cluster organization and from

Hox sequences shared across ambulacraria (including theptychoderid hemichordate Balanoglossus simodensis [23]and the echinoderms Metacrinus rotundus and Heliocidaristuberculata, which possess the same ambulacrarian-specificposterior Hox genes [Figure S3]), we suggest in Figure 3 (hypo-thetical cluster 1) that the ambulacrarian ancestor possessedminimally a 12-gene Hox cluster similar to the hemichordatecluster, with at least nine genes (hox1–hox9/10) arranged in agenomic order and transcriptional orientation the same as theirchordate orthologs. Of the three remaining posterior genes,one (11/13a;ambPa) was probably in tandem order and orien-tation with the hox1–hox9/10 genes, and two (hox11/13c andhox11/13b; ambPb and ambPc) were inverted at the 50 end ofthe ancestral cluster, like the posterior genes of extant hemi-chordate clusters (and like hox11/13a and hox11/13c ofS. purpuratus). Alternatively, as diagrammed in Figure 3 (hypo-thetical cluster 2), the ambulacrarian ancestor may have hadthe hox11/13b and hox11/13c genes, not inverted, but intandem with all other genes of the cluster, and then the11/13b,c genes inverted as a pair in the hemichordate lineagewhereas only 11/13 b inverted in the echinoderm lineage (withthe same rearrangements and gene loss for hox1–hox11/13ain the echinoderm lineage as described above). Furthermore,

Genomic Clustering of Hemichordate Hox Genes2057

in light of the large number of chordate posterior Hox genes,the ambulacrarian ancestor may have possessed more thanfour, with parallel gene loss in the hemichordate and echino-derm lineages or in their common stem lineage before theancestor (see Figure S4). Further elucidation of the ancestralcluster requires more information about hox sequences andcluster organization from additional ambulacraria.

From shared features of the chordate and ambulacrarianclusters, we suggest, as have others [15, 19, 20], that the lastcommon deuterostome ancestor had a well ordered clusterof at least nine genes including a hox9-like gene (hox9/10 inambulacraria). The number and kind of additional posteriorgenes of this ancestor remain difficult to estimate due to thediffering numbers of such genes in different extant lineages(seven in Amphioxus, six in vertebrates, four in Ambulacraria,and one or two in protostome outgroups) and their differinghomeodomain sequences, yielding only weak supportedorthologies by phylogenetic analysis. Sequence differencesof the posterior genes in deuterostome evolution have beenattributed to multiple gene duplications [19], gene loss, anddeuterostome posterior flexibility (extensive homeoboxsequence change [20]).

We join others [6, 19, 20, 24] in drawing attention to theextensive evolutionary modifications of posterior Hox genesin deuterostomes, far more than has occurred for their anteriorand central genes. This greater gene evolution at the 50 endof the cluster raises questions of whether significant Hox-related morphological evolution occurred at the posteriorend of the body axis of early deuterostomes, where thesegenes were presumably first expressed (although co-optionof themmay have occurred later for other uses). In extant chor-dates, they are expressed in the posterior body including thepostanal tail, a phylotypic trait of chordates (fin and limbexpression arising later in fish and tetrapod vertebrates), andin S. kowalevskii they are expressed in the posterior bodyand the contractile, adhesive postanal extension (sometimescalled ‘‘tail-like’’ or ‘‘stalk-like’’) of the juvenile animal [16, 17].(This postanal region is, however, absent in extant ptychoder-ids such as P. flava.) Among other deuterostomes (not yetanalyzed genomically), pterobranch hemichordates possessa contractile stalk (separated from the anus), crinoid echino-derms have stalks, and echinoderm-related fossils (e.g.,solutes) reveal muscular stalks/holdfasts [25]. We favor thepossibility that the deuterostome ancestor, and then earlystem Ambulacraria and chordates, engaged in posterior axialinnovations such as tails, stalks, and holdfasts, some withdorsoventral asymmetry to exclude the gut (hence, postanal),and that the evolution of these diverse modifications involvedthe expansion, loss, and sequence diversification of posteriorHox genes in the various lineages (Figure S4).

In summary, we find that two hemichordates, separatedby an estimated 400 MYa [14], have the same genomic organi-zation of their 12-member Hox clusters, and that apart fromdifferences at the posterior end of this cluster, the hemichor-date arrangementmatches that of amphioxus and vertebrates.Thus, these hemichordates should provide us with useful newsystems in which to address the function and regulation ofHox genes and gain greater insight into the ancestral deutero-stome condition.

Accession Numbers

cDNA and genomic sequences described in the present study were depos-

ited in GenBank and JDBD. NCBI accession numbers for the three

S. kowalevskii scaffolds containing Hox gene sequences from the Skow_1.1

assembly by the Baylor College of Medicine Human Genome Sequencing

Center are: Skow_1.1 scaffold1507NW_003106599;Skow_1.1 scaffold16417

NW_003121426; and Skow_1.1 scaffold26913 NW_003131859). The

HudsonAlpha assembly scaffold_166 has been deposited as GenBank

JX899680. The noncoding transcripts of the S. kowalevskii Hox cluster are

deposited as GenBank JX860400–JX86403.

Supplemental Information

Supplemental Information includes four figures, two tables, and Supple-

mental Experimental Procedures and can be found with this article online

at http://dx.doi.org/10.1016/j.cub.2012.08.052.

Acknowledgments

We thank the Baylor College of Medicine Human Genome Sequencing

Center for providing prepublication access to the transcriptome data and

the S. kowalevskii genome assembly, and we thank the HudsonAlpha

Institute of Biotechnology for prepublication access to the S. kowalevskii

assembly. This work is funded by NIH USPHS grants HD42724 to J.G. and

HD37277 to M.K., and by the National Science Foundation, Developmental

Mechanisms grant 0818679 to C.L., by Grants-in-Aids ofMEXT (17018018 to

N.S.) and JSPS (23570266 to T.I.), and by the Grant-in-Aid for Scientific

Research on Innovative Area ‘‘Genome Science’’ from MEXT Japan.

Received: June 1, 2012

Revised: August 9, 2012

Accepted: August 29, 2012

Published online: October 11, 2012

References

1. Delsuc, F., Brinkmann, H., Chourrout, D., and Philippe, H. (2006).

Tunicates and not cephalochordates are the closest living relatives of

vertebrates. Nature 439, 965–968.

2. Putnam, N.H., Butts, T., Ferrier, D.E., Furlong, R.F., Hellsten, U.,

Kawashima, T., Robinson-Rechavi, M., Shoguchi, E., Terry, A., Yu,

J.K., et al. (2008). The amphioxus genome and the evolution of the

chordate karyotype. Nature 453, 1064–1071.

3. Satoh, N. (2008). An aboral-dorsalization hypothesis for chordate origin.

Genesis 46, 614–622.

4. Gerhart, J., Lowe, C., and Kirschner, M. (2005). Hemichordates and the

origin of chordates. Curr. Opin. Genet. Dev. 15, 461–467.

5. Swalla, B.J., and Smith, A.B. (2008). Deciphering deuterostome

phylogeny: molecular, morphological and palaeontological perspec-

tives. Philos. Trans. R. Soc. Lond. B Biol. Sci. 363, 1557–1568.

6. Garcia-Fernandez, J. (2005). The genesis and evolution of homeobox

gene clusters. Nat. Rev. Genet. 6, 881–892.

7. Pearson, J.C., Lemons, D., and McGinnis, W. (2005). Modulating Hox

gene functions during animal body patterning. Nat. Rev. Genet. 6,

893–904.

8. Duboule, D. (2007). The rise and fall of Hox gene clusters. Development

134, 2549–2560.

9. Cameron, R.A., Rowen, L., Nesbitt, R., Bloom, S., Rast, J.P., Berney, K.,

Arenas-Mena, C., Martinez, P., Lucas, S., Richardson, P.M., et al. (2006).

Unusual gene order and organization of the sea urchin hox cluster.

J. Exp. Zoolog. B Mol. Dev. Evol. 306, 45–58.

10. Holland, L.Z., Albalat, R., Azumi, K., Benito-Gutierrez, E., Blow, M.J.,

Bronner-Fraser, M., Brunet, F., Butts, T., Candiani, S., Dishaw, L.J.,

et al. (2008). The amphioxus genome illuminates vertebrate origins

and cephalochordate biology. Genome Res. 18, 1100–1111.

11. Tagawa, K., Satoh, N., and Humphreys, T. (2001). Molecular studies of

hemichordate development: a key to understanding the evolution of

bilateral animals and chordates. Evol. Dev. 3, 443–454.

12. Rottinger, E., and Lowe, C.J. (2012). Evolutionary crossroads in devel-

opmental biology: hemichordates. Development 139, 2463–2475.

13. Peterson, K.J., Lyons, J.B., Nowak, K.S., Takacs, C.M., Wargo, M.J.,

and McPeek, M.A. (2004). Estimating metazoan divergence times with

a molecular clock. Proc. Natl. Acad. Sci. USA 101, 6536–6541.

14. Cannon, J.T., Rychel, A.L., Eccleston, H., Halanych, K.M., and Swalla,

B.J. (2009). Molecular phylogeny of hemichordata, with updated status

of deep-sea enteropneusts. Mol. Phylogenet. Evol. 52, 17–24.

Current Biology Vol 22 No 212058

15. Martinez, P., Rast, J.P., Arenas-Mena, C., and Davidson, E.H. (1999).

Organization of an echinoderm Hox gene cluster. Proc. Natl. Acad.

Sci. USA 96, 1469–1474.

16. Lowe, C.J., Wu, M., Salic, A., Evans, L., Lander, E., Stange-Thomann, N.,

Gruber, C.E., Gerhart, J., and Kirschner, M. (2003). Anteroposterior

patterning in hemichordates and the origins of the chordate nervous

system. Cell 113, 853–865.

17. Aronowicz, J., and Lowe, C.J. (2006). Hox gene expression in the hemi-

chordate Saccoglossus kowalevskii and the evolution of deuterostome

nervous systems. Integr. Comp. Biol. 46, 890–901.

18. Peterson, K.J. (2004). Isolation of Hox and Parahox genes in the hemi-

chordate Ptychodera flava and the evolution of deuterostome Hox

genes. Mol. Phylogenet. Evol. 31, 1208–1215.

19. Thomas-Chollier, M., Ledent, V., Leyns, L., and Vervoort, M. (2010). A

non-tree-based comprehensive study of metazoan Hox and ParaHox

genes prompts new insights into their origin and evolution. BMC Evol.

Biol. 10, 73.

20. Ferrier, D.E., Minguillon, C., Holland, P.W., and Garcia-Fernandez, J.

(2000). The amphioxus Hox cluster: deuterostome posterior flexibility

and Hox14. Evol. Dev. 2, 284–293.

21. Hara, Y., Yamaguchi, M., Akasaka, K., Nakano, H., Nonaka, M., and

Amemiya, S. (2006). Expression patterns of Hox genes in larvae of the

sea lily Metacrinus rotundus. Dev. Genes Evol. 216, 797–809.

22. Long, S., Martinez, P., Chen, W.C., Thorndyke, M., and Byrne, M. (2003).

Evolution of echinoderms may not have required modification of the

ancestral deuterostome HOX gene cluster: first report of PG4 and

PG5 Hox orthologues in echinoderms. Dev. Genes Evol. 213, 573–576.

23. Ikuta, T., Miyamoto, N., Saito, Y., Wada, H., Satoh, N., and Saiga, H.

(2009). Ambulacrarian prototypical Hox and ParaHox gene comple-

ments of the indirect-developing hemichordate Balanoglossus

simodensis. Dev. Genes Evol. 219, 383–389.

24. Monteiro, A.S., and Ferrier, D.E.K. (2006). Hox genes are not always

colinear. Int. J. Biol. Sci. 2, 95–103.

25. Smith, A.B. (2008). Deuterostomes in a twist: the origins of a radical

new body plan. Evol. Dev. 10, 493–503.