Identical Genomic Organization of Two Hemichordate Hox Clusters

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provided by Elsevier - Publisher Connector 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 Report Identical Genomic Organization of Two Hemichordate Hox Clusters

Robert Freeman,1,12 Tetsuro Ikuta,2,12 Michael Wu,3 [10] have similar organization to the hemichordate cluster, Ryo Koyanagi,2 Takeshi Kawashima,2 Kunifumi Tagawa,4 but with different posterior genes. These results provide Tom Humphreys,5 Guang-Chen Fang,6 Asao Fujiyama,7 genomic evidence for a well-ordered complex in the deutero- Hidetoshi Saiga,8 Christopher Lowe,9 Kim Worley,10 stome ancestor for the hox1–hox9/10 region, with the Jerry Jenkins,11 Jeremy Schmutz,11 Marc Kirschner,1 number and kind of posterior genes still to be elucidated. Daniel Rokhsar,3 Nori Satoh,2,* and John Gerhart3,* 1 Department of Systems Biology, Harvard Medical School, Results and Discussion Boston, MA 02114, USA 2 Marine Genomics Unit, Okinawa Institute of Science and Here we characterize the order, transcriptional orientation, and Technology Graduate University, Onna, clustering of the Hox genes of the genomes of two widely Okinawa 904-0495, Japan studied model hemichordates, Saccoglossus kowalevskii 3 Department of Molecular and Cell Biology, University of and Ptychodera ﬂava [4, 11, 12], that represent two major California, Berkeley, Berkeley, CA 94720-3200, USA evolutionary branches of enteropneust hemichordates that 4 Marine Biological Laboratory, Graduate School of Science, diverged an estimated 400 million years ago (MYa) [13]: Hiroshima University, Onomichi, Hiroshima 722-0073, Japan Saccoglossus, of the direct developing harimaniids, and 5 Department of Cell and Molecular Biology, University of Ptychodera, of the indirect developing ptychoderids [14]. Hawaii, HI 96822, USA Both are vermiform marine animals with gill slits, a nonseg- 6 Clemson University Genomics Institute, Clemson, mented body, and a muscular proboscis for burrowing (so- SC 29634, USA called ‘‘acorn worms’’). A third branch, not included here, is 7 National Institute of Genetics, Mishima, that of pterobranch hemichordates, which may be an offshoot Shizuoka 411-8540, Japan of the harimaniids or basal to both branches [14]. Together, the 8 Department of Biological Sciences, Graduate Schools of phyla of hemichordates and echinoderms (diverged an esti- Science and Engineering, Tokyo Metropolitan University, mated 540 MYa [13]) comprise the Ambulacraria, sharing Hachiohji, Tokyo 192-0397, Japan a last common ambulacrarian ancestor. Similarly, the Ambula- 9 Department of Biology, Stanford University, Hopkins Marine craria and the chordate phylum comprise the Deuterostome Station, Paciﬁc Grove, CA 93950, USA superclade, sharing a last common deuterostome ancestor. 10 Department of Molecular and Human Genetics, Human The similarities of Hox genes and their clusters across these Genome Sequencing Center, Baylor College of Medicine, groups can, if found, provide information about the Hox Houston, TX 77030, USA complement of the ambulacrarian and deuterostome ances- 11 HudsonAlpha Institute for Biotechnology, 601 Genome Way, tors that gave rise to these three diverse phyla [3–5]. Huntsville, AL 35806, USA

Characterization of the Hox Genes of S. kowalevskii Summary and P. flava Following the isolation of ANTP-class Hox gene sequences Genomic comparisons of chordates, hemichordates, and from the sea urchin Strongylocentrotus purpuratus [15], echinoderms can inform hypotheses for the evolution of an echinoderm, complementary DNA (cDNA) clones were these strikingly different phyla from the last common obtained from hemichordates for 11 of 12 Hox genes in deuterostome ancestor [1–5]. Because hox genes play S. kowalevskii (excepting hox8)[16, 17], and 8 of 12 in pivotal developmental roles in bilaterian animals [6–8], we P. flava (excepting hox2, hox3, hox7, and hox8)[18]. At the analyzed the Hox complexes of two hemichordate genomes. time, there was no information on the genomic linkage of We find that Saccoglossus kowalevskii and Ptychodera flava Hox genes in hemichordates. For hemichordate genes hox1– both possess 12-gene clusters, with mir10 between hox4 hox8, numerical identities were preliminarily assigned based and hox5, in 550 kb and 452 kb intervals, respectively. Genes on the best match of the homeodomain sequence to paralo- hox1–hox9/10 of the clusters are in the same genomic order gous groups of vertebrate Hox genes and to the sea urchin and transcriptional orientation as their orthologs in chor- genes. Hox9/10 and hox11/13a,b,c, which were first named dates, with hox1 at the 30 end of the cluster. At the 50 end, in S. purpuratus [15] because of their broad similarity to each cluster contains three posterior genes specific to vertebrate posterior genes, were used as gene names for Ambulacraria (the hemichordate-echinoderm clade), two subsequently discovered ambulacrarian orthologs. The hox1– forming an inverted terminal pair. In contrast, the echino- hox11/13c (except for hox8) genes of S. kowalevskii are derm Strongylocentrotus purpuratus contains a 588 kb expressed along the anteroposterior axis of embryonic ecto- cluster [9] of 11 orthologs of the hemichordate genes, derm and juvenile epidermis [16, 17] in the same order as their ordered differently, plausibly reflecting rearrangements of assigned numerical gene names, and similar to the ordered an ancestral hemichordate-like ambulacrarian cluster. Hox expression of the orthologous Hox genes in the vertebrate clusters of vertebrates and the basal chordate amphioxus central nervous system and in the ectoderm of protostomes such as Drosophila [6, 7]. In the course of our genomic analysis, we obtained the 12These authors contributed equally to this work previously uncharacterized Hox genes of S. kowalevskii and *Correspondence: [email protected] (N.S.), [email protected] (J.G.) P. flava (see Supplemental Experimental Procedures available Current Biology Vol 22 No 21 2054

online). The total complements of 12 genes each for these species are summarized in the phylogenetic tree of Figure 1 and the homeodomain sequence alignments of Figures S1 and S2. Membership of each gene in a vertebrate-defined paralogous group was further assessed by submitting the homeodomain sequences to HoxPred [19]. From these analyses, hox1–hox8 of the two hemichordates are found to strongly resemble each other as well as hox genes of echinoderms and chordates. The hemichordate sets contain: (1) two anterior genes (hox1 and hox2) probably descended from PG1 and PG2 orthologs dating back to the bilaterian ancestor, (2) a group3 gene (hox3) probably descended from a PG3 ortholog of that ancestor, and (3) five central genes (hox4, hox5, hox6, hox7, hox8) that can be matched with chordate counterparts. The four posterior genes (hox9/10, hox11/13a,b,c) of the hemichordate sets, although clearly similar to each other and to the echinoderm posterior genes and although having PG9 character, have only poorly supported phylogenetic relation- ships to specific chordate posterior genes, as others have noted [15, 20]. From phylogenetic analysis of the 60 aa home- odomains (Figure 1) as well as of 75 aa blocks encompassing the homeodomain and conserved among posterior hox genes (Figure S3), we find that the hemichordate and echinoderm hox9/10 genes, in keeping with their name, group with vertebrate 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 group separate from those of vertebrate hox11–hox14 and amphioxus hox13–15, and appear to have diverged within the ambulacrarian lineage (Figures S2 and S3). Lacking evidence for orthology to vertebrate hox11, hox12, hox13 , and with evidence for their close-relatedness to the echinoderm genes, we suggest that the 11/13a,b,c genes be renamed as ambPa,b,c (ambulacrarian Posterior a,b,c). The extensive diversification of posterior gene sets in the different lineages of deuterostomes, as compared to their anterior and central gene sets, has been attributed to ‘‘deuterostome posterior flexibility’’ [20] and to multiple independent duplications [19], as discussed later.

Assembly of the Hox Complexes For both hemichordates, the genomic analysis entailed the isolation of overlapping BAC sequences carrying subsets of hox genes, complemented by the assembly of whole genome shotgun 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-gene cluster. Nine genes (hox1 to hox9/10) are arranged in the same order and transcriptional orientation as their vertebrate orthologs, with hox1 at the 30end, and with three posterior Figure 1. Molecular Phylogenetics of the Hemichordate Hox Genes genes at the 50 end, namely, hox11/13a in tandem with hox1– A maximum-likelihood phylogram was constructed from the 60-amino-acid 9/10 but hox11/13c and 11/13b inverted as a terminal pair. homeodomain sequences from 64 Hox genes, and using two NK2.1 genes as The entire cluster, from exon2 of hox1 to exon2 of the inversely an outgroup. The Hox genes of the hemichordates P. flava and S. kowalevskii are shown in blue. Red circles indicate genes characterized anew in this oriented hox11/13b, spans a genomic distance of 550 kb. study. PhyML was used (www.phylogeny.fr/version2_cgi/one_task.cgi? A hox8-like homeodomain sequence was recovered in the task_type=phyml; see Supplemental Experimental Procedures). The aLRT interval between hox7 and hox9/10 (deposited as a GNOMON branch support values are shown as percentage in red. Branches with model [GenBank GI291221533]). Furthermore, a mir10 se- support values under 50% were collapsed. Branch length is indicated by quence was found between hox4 and hox5. the bar in the lower right. Pf, Ptychodera flava; Sk, Saccoglossus kowalevskii; For P. flava, four BAC clones and three genome shotgun- Sp, Strongylocentrotus purpuratus;Mr,Metacrinus rotundus (for hox4, which is absent in S. purpuratus); Am, Branchiostoma floridae (amphioxus); derived contigs were linked to form a supercontig of 455.7 kb, and Mm, Mus musculus. which included the 12 P. flava Hox genes within a 451.7 kb- See also Figures S1 and S2 and Table S1. long genomic region (Figure 2B). Remarkably, the organization Genomic Clustering of Hemichordate Hox Genes 2055

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. of the cluster is the same as that of S. kowalevskii, namely, hox1 For the P. flava complex, all genes have two exons except to hox9/10 are arranged in the same order and transcriptional hox5 and hox7, which have three, and all homeobox se- orientation as their vertebrate orthologs, with hox1 at the quences are contained in single exons (the estimates of 30end, and three posterior genes are arranged at the 50 end, different numbers of exons for hox5, hox7, and hox9 between with hox11/13a in tandem with hox1-9/10 but hox11/13c and the two species are not definitive because they are based on hox11/13b inverted as a terminal pair. A mir10 sequence cDNA analysis in some cases and on gene prediction in others; resides between hox4 and hox5. Tables S1A and S1B). The average mRNA size is 1,691 nucle- From the assembled complexes we estimated the exon- otides in the P. flava complex. The average intergenic distance intron structure, size, and intergenic distances of the 12 genes is 34 kb for all genes of the cluster. Like S. kowalevskii, the of each (Tables S1A and S1B). For S. kowalevskii, all genes have largest (66 kb) intergenic space occurs between the group of two exons except hox9/10, which has four, and all homeobox ten tandem genes (hox1–hox11/13a) and the inverted pair sequences are contained within single exons. The average (hox11/13c and hox11/13b). primary transcript size and messenger RNA (mRNA) size for In both complexes, no other protein-coding gene was iden- the 12 genes are 5,698 and 2,651 nucleotides, respectively tified within the cluster by routine sequence searches (Figures (based on cDNA sequences placed against the Scaffold_166 2A and 2B). However, for S. kowalevskii, two sense and two sequence; Supplemental Experimental Procedures). An inter- antisense noncoding transcripts of unknown function have genic distance of 127 kb separates hox11/13a and hox11/ been detected in and near the cluster (Table S2; Supplemental 13c; that is, it separates the group of ten tandemly ordered Experimental Procedures). Outside the S. kowalevskii cluster, genes (hox1–hox11/13a) from the inverted pair (hox11/13c an astacin-like protease sequence and a thioredoxin4-like and hox11/13b). When this interval is omitted, the average in- sequence reside within 11 kb and 47 kb, respectively, of the tergenic distance is 36 kb for the remaining genes of the cluster. terminal hox11/13b gene. However, within the 110 kb interval Current Biology Vol 22 No 21 2056

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 ambulacrarian 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.

from hox11/13b to the end of Scaffold_166, no evx sequence hox5 to the 50 end of the cluster, followed by loss of hox4 was found, whereas evx is located adjacent to posterior genes but retention of mir10, plus an inversion and translocation of of vertebrate clusters A and D, the amphioxus cluster, and the hox11/13c, the outcome would be the present-day echino- sea urchin cluster. The S. kowalevskii evx gene is on a separate derm S. purpuratus cluster of mixed orders and orientations. Scaffold_145 (954 kb), 120 kb from one end; we have no The loss of hox4 presumably occurred after the echinoid evidence linking this scaffold to that of the Hox cluster. lineage split from crinoids and asteroids, which have hox4 In summary, these two hemichordate species, separated [21, 22]. This and other rearrangement schemes have been by an estimated 400 MYa [14], possess 12-gene Hox clusters previously discussed [9, 15]. of identical organization, with nine genes (hox1–hox9/10) From the comparisons of cluster organization and from arranged in the same genomic order and transcriptional orien- Hox sequences shared across ambulacraria (including the tation as their orthologs in chordates, with hox1 at the 30 end ptychoderid hemichordate Balanoglossus simodensis [23] of the cluster. At the 50 end, both clusters contain three poste- and the echinoderms Metacrinus rotundus and Heliocidaris rior genes specific to Ambulacraria, two forming an inverted tuberculata, which possess the same ambulacrarian-specific terminal pair. The cluster sizes are 550 and 452 kb, respec- posterior Hox genes [Figure S3]), we suggest in Figure 3 (hypo- tively, for S. kowalevskii and P. flava. thetical cluster 1) that the ambulacrarian ancestor possessed minimally a 12-gene Hox cluster similar to the hemichordate Comparison of Deuterostome Hox Clusters cluster, with at least nine genes (hox1–hox9/10) arranged in a Figure 3 summarizes the genomic Hox clusters for the two genomic order and transcriptional orientation the same as their hemichordates reported here and for the sole echinoderm chordate orthologs. Of the three remaining posterior genes, example, the 11-gene cluster of S. purpuratus, which has a one (11/13a;ambPa) was probably in tandem order and orien- different order and orientation of genes from the hemichordate tation with the hox1–hox9/10 genes, and two (hox11/13c and clusters. These differences can be interpreted as arising by hox11/13b; ambPb and ambPc) were inverted at the 50 end of way of rearrangements of an ancestral ambulacrarian cluster the ancestral cluster, like the posterior genes of extant hemi- having hemichordate-like organization (Figure 3, hypothetical chordate clusters (and like hox11/13a and hox11/13c of cluster 1). That is, five genes in the middle of the echinoderm S. purpuratus). Alternatively, as diagrammed in Figure 3 (hypo- cluster (hox6, hox7, hox8, hox9/10, hox11/13a) have the thetical cluster 2), the ambulacrarian ancestor may have had same order and orientation as their orthologs in the hemichor- the hox11/13b and hox11/13c genes, not inverted, but in date clusters (Figure 3; dotted lines). If within the lineage to sea tandem with all other genes of the cluster, and then the urchins, the ancestral ambulacrarian cluster underwent an 11/13b,c genes inverted as a pair in the hemichordate lineage inversion of the (hox1, hox2, hox3, hox4, mir10, hox5) group whereas only 11/13 b inverted in the echinoderm lineage (with of genes at the 30 end of the cluster, and then a translocation the same rearrangements and gene loss for hox1–hox11/13a of the inverted genes (hox1, hox2, hox3, hox4, mir10) but not in the echinoderm lineage as described above). Furthermore, Genomic Clustering of Hemichordate Hox Genes 2057

in light of the large number of chordate posterior Hox genes, S. kowalevskii scaffolds containing Hox gene sequences from the Skow_1.1 the ambulacrarian ancestor may have possessed more than assembly by the Baylor College of Medicine Human Genome Sequencing four, with parallel gene loss in the hemichordate and echino- Center are: Skow_1.1 scaffold1507 NW_003106599; Skow_1.1 scaffold16417 NW_003121426; and Skow_1.1 scaffold26913 NW_003131859). The derm lineages or in their common stem lineage before the HudsonAlpha assembly scaffold_166 has been deposited as GenBank ancestor (see Figure S4). Further elucidation of the ancestral JX899680. The noncoding transcripts of the S. kowalevskii Hox cluster are cluster requires more information about hox sequences and deposited as GenBank JX860400–JX86403. cluster organization from additional ambulacraria. From shared features of the chordate and ambulacrarian Supplemental Information clusters, we suggest, as have others [15, 19, 20], that the last common deuterostome ancestor had a well ordered cluster Supplemental Information includes four figures, two tables, and Supple- of at least nine genes including a hox9-like gene (hox9/10 in mental Experimental Procedures and can be found with this article online at http://dx.doi.org/10.1016/j.cub.2012.08.052. ambulacraria). The number and kind of additional posterior genes of this ancestor remain difficult to estimate due to the Acknowledgments differing numbers of such genes in different extant lineages (seven in Amphioxus, six in vertebrates, four in Ambulacraria, We thank the Baylor College of Medicine Human Genome Sequencing and one or two in protostome outgroups) and their differing Center for providing prepublication access to the transcriptome data and homeodomain sequences, yielding only weak supported the S. kowalevskii genome assembly, and we thank the HudsonAlpha orthologies by phylogenetic analysis. Sequence differences Institute of Biotechnology for prepublication access to the S. kowalevskii of the posterior genes in deuterostome evolution have been 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 attributed to multiple gene duplications [19], gene loss, and Mechanisms grant 0818679 to C.L., by Grants-in-Aids of MEXT (17018018 to deuterostome posterior flexibility (extensive homeobox N.S.) and JSPS (23570266 to T.I.), and by the Grant-in-Aid for Scientific sequence change [20]). Research on Innovative Area ‘‘Genome Science’’ from MEXT Japan. We join others [6, 19, 20, 24] in drawing attention to the extensive evolutionary modifications of posterior Hox genes Received: June 1, 2012 in deuterostomes, far more than has occurred for their anterior Revised: August 9, 2012 and central genes. 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