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Developmental and Comparative Immunology 35 (2011) 705–715

Contents lists available at ScienceDirect

Developmental and Comparative Immunology

journa l homepage: www.e lsev ier .com/ locate /dc i

eview

family tree of vertebrate chemokine receptors for a unified nomenclature

isayuki Nomiyamaa,∗, Naoki Osadab, Osamu Yoshiec

Department of Molecular Enzymology, Kumamoto University Faculty of Life Sciences, Honjo, Kumamoto 860-8556, JapanDivision of Evolutionary Genetics, Department of Population Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540, JapanDepartment of Microbiology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka 589-8511, Japan

r t i c l e i n f o

rticle history:eceived 23 November 2010eceived in revised form 25 January 2011ccepted 25 January 2011

a b s t r a c t

Chemokines receptors are involved in the recruitment of various cell types in inflammatory and physi-ological conditions. There are 23 known chemokine receptor genes in the human genome. However, itis still unclear how many chemokine receptors exist in the genomes of various vertebrate species otherthan human and mouse. Moreover, the orthologous relationships are often obscure between the genes

vailable online 2 February 2011

eywords:hemokine receptorsene clustervolution

of higher and lower vertebrates. In order to provide a basis for a unified nomenclature system of thevertebrate chemokine receptor gene family, we have analysed the chemokine receptor genes from thegenomes of 16 vertebrate species, and classify them into 29 orthologous groups using phylogenetic andcomparative genomic analyses. The results reveal a continuous gene birth and death process during thevertebrate evolution and an interesting evolutionary history of the chemokine receptor genes after the

ene duplicationertebrates

emergence in agnathans.© 2011 Elsevier Ltd. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7052. Sequence and genomic data of vertebrate chemokine receptor genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7063. Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7064. Mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 707

4.1. Human and mouse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7074.2. Other mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 707

5. Birds, lizard, and frog . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7086. Jawed and jawless fishes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7107. Evolutionary history of the chemokine receptor genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7138. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 714Appendix A. Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 714References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 714

. Introduction

Chemokines are a multigene family of small, secreted cytokinesediating cell migration during inflammation, immune surveil-

ance, and organogenesis (Moser et al., 2004; Zlotnik and Yoshie,

three amino acids are inserted between the first two of the fourcysteine residues. In the CC subfamily, the first two cysteines arearranged in adjacent positions. The first and third cysteine residuesare absent in the XC (or C) subfamily with only one disulfide bond.

000). Chemokines are subdivided into 5 subfamilies (CXC, CC, XC,X3C, and CX) based on the arrangement of four conserved cysteineesidues involved in the formation of disulfide bonds (Nomiyamat al., 2010). In the CXC and CX3C chemokine subfamily, one or

∗ Corresponding author. Tel.: +81 96 373 5065; fax: +81 96 373 5066.E-mail address: nomiyama@gpo.kumamoto-u.ac.jp (H. Nomiyama).

145-305X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.dci.2011.01.019

Furthermore, we have recently described yet another subfamily CXfrom the analysis of Zebrafish chemokine genes (Nomiyama et al.,2008). The CX subfamily lacks one of the first two cysteine residuesbut not the third one.

Besides the structural criteria, chemokines can be categorized

into several groups from the functional point of view (Mantovaniet al., 2006; Nomiyama et al., 2010). Inflammatory chemokines arethose upregulated in inflammatory conditions and involved in therobust recruitment of leukocytes to inflamed sites. Homeostatic

7 Comp

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06 H. Nomiyama et al. / Developmental and

hemokines are those produced constitutively at non-inflamedites and involved in homeostatic migration and homing of cellsn physiological conditions. Some chemokines have both proper-ies, and are thus called dual-function chemokines. In addition,ome chemokines are constitutively expressed at high levels andre present in the blood stream as inactive forms (Berahovich et al.,005; Mortier et al., 2008) or stored in alpha-granules of plateletsBrandt et al., 2000). The plasma chemokines can be activated byroteolytic cleavage at inflamed sites, while platelet chemokinesre released upon platelet activation. Thus, they could be consid-red as a kind of inflammatory chemokines.

To date, chemokines have only been described in vertebratesBajoghli et al., 2009; DeVries et al., 2006). The human genome con-ains more than 44 members (Zlotnik et al., 2006). The chemokineenes have been evolved rapidly, both in their sequences and theize of gene family (Nomiyama et al., 2010). Thus, the orthologouselationships of some chemokines between the species could benclear. Especially the chemokines between fishes and other ver-ebrates are highly divergent (Nomiyama et al., 2008).

All the known chemokine receptors (ChemRs) are seven trans-embrane domain G protein-coupled receptors (GPCRs) that

ommonly couple to the G�i class of the heterotrimeric G proteins.hemRs are classified according to the subfamily of chemokine lig-nds that they bind; CXCR, CCR, XCR, and CX3CR. It is still unknownhether there is a specific group of receptors for CX chemokines

n zebrafish (Nomiyama et al., 2008). So far, 18 genes encodinghemRs with standard chemotactic functions have been identified

n the human genome: 6 CXCR genes, 10 CCR genes, 1 XCR gene,nd 1 CX3CR gene. ChemRs often bind more than one chemokine,hile a single chemokine often binds to more than one receptor.

his binding promiscuity is one of the characteristic features of thehemokine system and is mostly observed between inflammatorynd plasma chemokines and their receptors (Mantovani, 1999). Inontrast, homeostatic chemokines and their receptors display morepecific relationships (Yoshie et al., 1997, 2001). Furthermore, 5enes encoding atypical (non-signaling) ChemRs have been iden-ified (DARC, CCBP2, CCRL1, CCRL2, CXCR7) (Graham, 2009; Leickt al., 2010; Naumann et al., 2010). These atypical ChemRs areften called ‘silent’ or ‘decoy’ receptors since they bind chemokinesut do not elicit standard chemotactic responses following lig-nd binding. They are considered to be the scavengers for excesshemokines. Although the functional property of CXCR7 is still con-roversial (Thelen and Thelen, 2008), it has been shown to act as acavenger receptor for CXCL12 and CXCL11 (Naumann et al., 2010).

Compared to the chemokine ligand genes, the ChemR genes areelatively well conserved across the vertebrate species. However,ecause of a fish-specific whole-genome duplication event, whichccurred in the stem lineage of ray-finned (actinopterygian) fishesincluding teleosts) after divergence from the land vertebratesMeyer and Van de Peer, 2005; Sato and Nishida, 2010), togetherith their higher rates of gene rearrangement and faster evolu-

ion of protein sequences compared to mammals, there occurredarge increases in the number of genes in the teleost fishes (Ravind Venkatesh, 2008). This makes it quite difficult to assign therthologous relationships for the members of multigene familiesetween teleost fishes and other vertebrates. Nevertheless, it maye still desirable to have a unified nomenclature system that coversll the vertebrate ChemR genes.

Several groups have identified ChemR genes from various ver-ebrate species using comparative genomics-based data miningDeVries et al., 2006; Kaiser et al., 2005; Liu et al., 2009; Wang et al.,

005). However, the draft genome sequences they used were theld versions, and the numbers of species investigated were lim-ted. To provide a more up-to-date view of the ChemR genes ofertebrates, we have made a census of the vertebrate ChemR genessing the latest versions of 16 representative vertebrate genomes

arative Immunology 35 (2011) 705–715

covering a wide range of animal groups; jawless fish (1 species),cartilaginous fish (1), teleost fish (3), amphibian (1), reptile (1), bird(3), and mammal (6). Previously, we have shown that a combina-tion of phylogenetic and synteny-based approaches using severalspecies is quite useful to resolve the evolutionary history of a genefamily (Nomiyama et al., 2010). In the present study, therefore, wehave chosen only those animals, whose genome-wide sequencingis in progress or has already been finished, and performed robustand through phylogenetic and genomic analyses. If available, wehave also used multiple species of the same animal group but notbelonging to the same order so that the obtained ChemR gene con-tent of each species complements each other and the combineddata represent the ChemR entity of the animal group. From suchanalyses, a plausible evolutionary history of ChemR genes couldbe drawn. Indeed, the obtained results show that continuous geneexpansion and contraction events occurred at different time pointsduring the vertebrate evolution. We have also classified the ver-tebrate ChemR genes into 29 orthologous groups, each of whichcontains 0–4 paralogous genes from one species.

2. Sequence and genomic data of vertebrate chemokinereceptor genes

We have identified most of the vertebrate ChemR genes inthe NCBI or Ensembl annotation. Supplementary Table 1 lists theIDs of their amino acid sequences. We have also identified someChemR genes by BLAST search. There is, however, a possibilitythat the genes predicted without cDNA sequences may includepseudogenes. In supplementary Fig. 1, we show the full aminoacid sequence of each gene with DRY (Rovati et al., 2007), CWLP(Shi et al., 2002), TXP (Govaerts et al., 2001), and NPxxY(x)5,6F(Fritze et al., 2003) motifs highlighted. The versions of the assem-bled genome sequences used are also shown in the figure. Insupplementary Fig. 2, we show the phylogenetic trees that wereconstructed using the neighbor-joining method with Dayhoff’s(PAM) matrix and removing gaps by pairwise deletion (Saitou andNei, 1987) (supplementary Fig. 2). We used the sequences corre-sponding to the ChemR domain pfam00001 (Finn et al., 2010) inthe tree construction to exclude the relatively non-conserved N-and C-terminal sequences (supplementary Fig. 3). The tree shownin (B) of supplementary Fig. 2 is constructed by excluding 4 incom-plete sequences, platypus CXCR6a and CXCR6b, Tetraodon CCR11a,and elephant shark CXCR4, since adding incomplete sequences inthe tree construction may obscure the orthology (Shields, 2003).In supplementary Fig. 4, we show the comparative genomic mapsdrawn by using Ensembl data.

3. Nomenclature

Genes of several vertebrate species, human, mouse, chicken,Xenopus, and zebrafish, have their own ‘official’ gene symbolsassigned by the respective nomenclature committees. As forthe human ChemRs, a systematic nomenclature was proposed(Murphy, 2002; Murphy et al., 2000), and the nomenclature com-mittee for human genome organization adopted this system, inwhich each receptor is identified by its ligand subfamily plus R(receptor) and a given identifying number. For example, CXCR1refers to CXC chemokine receptor 1, whose representative commonname is IL8R�. In this review, we propose to categorize vertebrateChemR genes into 29 orthologous groups based on both the phy-

logenetic trees and genomic synteny. The orthologous groups arebasically defined by the human ChemR members, and thus thename of each group is based on the representative human gene.Each orthologous group includes a true ortholog having evolvedfrom a common ancestor and their paralogs having arisen from

Comparative Immunology 35 (2011) 705–715 707

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Table 1Human and mouse chemokine receptor genes.

Chemokine receptorname (human genesymbola)

Mouse genesymbola

Other name Number of AA(human)

CXCRCXCR1 Cxcr1 IL8RA 350CXCR2 Cxcr2 IL8RB 360CXCR2P1 – (pseudogene) –CXCR3 Cxcr3 GPR9 415,368,267CXCR4 Cxcr4 LESTR 356, 352CXCR5 Cxcr5 BLR1 327, 372CXCR6 Cxcr6 BONZO 342

CCRCCR1 Ccr1 CC-CKR-1 355– Ccr1l1 CC-CKR-1-likel (356)b

CCR2 Ccr2 CC-CKR-2 360, 374CCR3 Ccr3 CC-CKR-3 355, 373, 376CCR4 Ccr4 CC-CKR-4 360CCR5 Ccr5 CC-CKR-5 352CCR6 Ccr6 STRL22 374CCR7 Ccr7 BLR2 378CCR8 Ccr8 GPR-CY6 355CCR9 Ccr9 GPR-9-6 357, 369CCR10 Ccr10 GPR2 362

XCRXCR1 Xcr1 GPR5 333

CX3CRCX3CR1 Cx3cr1 V28 355, 362, 387

AtypicalCXCR7 Cxcr7 RDC1 362CCBP2 Ccbp2 D6 384CCRL1 Ccrl1 CCX-CKR 350CCRL2 Ccrl2 HCR 344, 356DARC Darc Duffy 338,336

H. Nomiyama et al. / Developmental and

pecies- or lineage-specific duplication events. In the case of groupsithout human genes, we have given new names: CCR11, CCR12,

nd CCR14. Since CCR13 is already used for a zebrafish gene, CCR13s not used for a group name.

The gene symbols of the species other than human are in gen-ral given the same names as their human orthologs. However,hen there are two or more paralogous genes resembling a sin-

le human gene, there is no unified rule to resolve such cases. Forxample, zebrafish gene symbols ccr6a and ccr6b are assigned toenes resembling human CCR6, while symbols ccr8.1 and ccr8.2 arellocated to two paralogous genes resembling human CCR8. In thiseview, we adhere to the gene symbols assigned by the commit-ees where appropriate, and alphabets in small letters are added toistinguish the genes in the same group, for example, CXCR1a andXCR1b.

In some cases, genes from different species with the same geneymbol are not orthologous. For example, Xenopus ccr3 gene waslassified into a newly defined CCR12 group by our analyses, and theene is thus named as CCR12 (ccr3) to show that the gene havinghe gene symbol ccr3 belongs to the group CCR12.

The names of the ChemR genes reported in the variousapers and the names used in this review are summarized inupplementary Table 2.

. Mammals

.1. Human and mouse

There are 23 known ChemR genes in the human genome, whilene additional gene termed Ccr1l1 (Ccr1-like 1) exists in the mouseenome (Table 1). Fig. 1 shows the genomic organization of theuman ChemRs and their chemokine ligands. The signaling recep-ors for CXCL14, CXCL17, and CCL18 are as yet unknown. There isne major gene cluster of ChemRs on the human chromosome 3.he cluster spans a large region of approximately 13 Mb in lengthontaining 12 genes encoding for CXCR6, CCR1 to 5, 8 and 9, XCR1,X3CR1, and two atypical receptors CCBP2 and CCRL2. Anothertypical receptor gene CCRL1 resides 86 Mb apart from the majorluster on the same human chromosome. Although most of theeceptors in the cluster interact with the inflammatory chemokines,he receptors CCR9, CXCR6, and XCR1 bind homeostatic or dual-unction chemokines and their genes are also arranged next toach other in the middle of the cluster. Phylogenetically, CCR9nd CXCR6 are closely related but are relatively distantly relatedrom the other ChemRs in the cluster (supplementary Fig. 2). TheX3CR1 gene resides between the genes for CCR4 and CCR8, andhey are also closely clustered in the tree (supplementary Fig. 2).he remaining ChemR genes are found as individual genes or inini-clusters (Fig. 1).Overall, the genomic organization of mouse ChemR genes is

uite similar to that of the human genes (supplementary Fig. 4).owever, mouse atypical ChemR genes Ccrl1 (Ccr-like-1) and Ccrl2re organized differently from the human counterparts. Whileuman CCRL2 gene is located at one end of the major cluster andCRL1 is located apart from the CCRL2 gene, both Ccrl1 and Ccrl2enes in the mouse genome, which are closely linked, are translo-ated to the opposite end of the cluster. Moreover, mouse Ccr1l1,hich is not present in the human genome, is located betweencr1 and Ccr3 in the major cluster. Ccr1l1 is closely related to Ccr1nd grouped in the CCR1 orthologous group together with Ccr1

Table 2). Although the Ccr1l1 is not characterized in detail (Gaond Murphy, 1995; Nibbs et al., 1997; Perelygin et al., 2008), it con-ains the DRY-like motif for the G protein coupling as well as thether ChemR motifs (supplementary Fig. 1), suggesting its signalingapacity.

a Human and mouse gene symbols and GeneIDs were taken from the web siteEntrezGene: http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene.

b Number of the amino acid sequence of the mouse gene.

Like chemokine ligands, ambiguous orthologous relationshipsare present for some ChemR genes in the clusters. ParalogousCXCR1 and CXCR2 genes in the CXCR mini-cluster have been shownto undergo gene conversion in the human and rabbit genomes(Shields, 2000). Evidence for gene conversions between CCR2 andCCR5 and between CCR1 and CCR3 has also been shown in a num-ber of mammals (Carmo et al., 2006; Esteves et al., 2007; Perelyginet al., 2008; Shields, 2000; Vazquez-Salat et al., 2007). Since geneconversion homogenizes the nucleotide sequences of paralogousgenes, the orthologous relationships could not be defined only byphylogenetic analysis (Shields, 2003).

Like chemokine ligands, ChemRs are known to form homo- orhetero-dimers (Thelen et al., 2010). Such dimer formation maymodulate the receptor activity, although the physiological rele-vance of the receptor oligomerization still largely remains unclear.However, it is plausible that gene conversion between adjacentgenes such as CCR1 and CCR3 would promote not only their shar-ing of ligands but also their heterodimer formation (Shields, 2000;Vazquez-Salat et al., 2007).

4.2. Other mammals

In addition to human and mouse, we have also analysed fourmammalian species: cow (Bos taurus, order Artiodactyla), dog(Canis familiaris, order Carnivora), opossum (Monodelphis domes-

tica, order Didelphimorphia, Infraclass Metatheria), and platypus(Ornithorhynchus anatinus, order Monotremata, subclass Protothe-ria). In contrast to the chemokine genes (Nomiyama et al., 2010),one-to-one orthologous relationships are mostly evident for theChemR genes between human and other mammals, and a highly

708 H. Nomiyama et al. / Developmental and Comparative Immunology 35 (2011) 705–715

CC

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6

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16

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222 3

19

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4

5

7

8

11

13

17

3

5

3L1

7

8

13

14

3

4

3L1

5

8

14

16

15

16

23

22

3L1

5

7

21

25

8

11

13

7

2

8

11

13

16

20

14

15

24

26

26

28

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2

5

7

11

13

14

17

23

24

17

CX

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CX

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2

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11

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6

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44

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9

10

11

19

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27

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CXCL CCL CX3CL XCL

receptorgene

ligand

receptorgene

ligand

inflammatorychemokine

dual-functionchemokine

homeostaticchemokine

plasma orplateletchemokine

F e cher g rece

cihrstCmlgFtgrhic

ig. 1. Genomic organization of the human chemokine receptors. Arrows indicate thecognized by each receptor are also shown below the receptor genes. The signalin

onserved synteny is maintained across the mammalian speciesnvestigated (supplementary Fig. 4). In the cow and dog genomes,owever, the major gene cluster is split into two. While the twoegions are located apart from each other on the same chromo-ome in the cow genome, they reside on separate chromosomes inhe dog genome. As seen in the mouse genome, the atypical ChemRsCRL1 and CCRL2 are closely located in the opossum and platypusajor clusters, strengthening that it was originally generated by a

ocal duplication in the major cluster. Furthermore, some ChemRenes may be still missing in the opossum and platypus genomes.or example, both opossum and platypus appear to lack CXCR3, butwo (CXCL9 and CXCL10) and one (CXCL10) of the CXCR3 ligand

enes have been identified in the opossum and platypus genomes,espectively (Nomiyama et al., 2010). Similarly, the CXCR5 geneas not been found in platypus, but its ligand CXCL13 gene exists

n the genome (Nomiyama et al., 2010). Several genes were dupli-ated in some species. CCR1L1 is found between CCR1 and CCR3

mokine receptor genes and their transcriptional orientation. The chemokine ligandsptors for CXCL14, CXCL17, and CCL18 are as yet unknown.

genes in the cow genome as in the mouse genome. The genes forCCR8 and CXCR6 were duplicated in the opossum and platypusgenomes, respectively. The platypus genome contains two genessimilar to human CXCR1 and CXCR2. It is however impossible todetermine which gene is orthologous to human CXCR1 or CXCR2from the genomic comparison due to the lack of markers aroundboth genes. The platypus genes are therefore tentatively designatedCXCR1a and CXCR1b.

5. Birds, lizard, and frog

Three species were selected from the birds for the analyses:

chicken (Gallus gallus, order Galliformes), zebra finch (Taeniopy-gia guttata, order Passeriformes), and duck (Anas platyrhynchos,order Anseriformes). Out of reptiles and amphibians, the genomesequencing of the anole lizard (Anolis carolinensis, order Squamata,class Reptilia) and Xenopus tropicalis (order Anula, class Amphibia)

H. Nomiyama et al. / Developmental and Comparative Immunology 35 (2011) 705–715 709

Table 2Chemokine receptor gene groups of mammalsa.

ChemR group Human Mouse Cow Dog Opossum Platypus

CXCR1 CXCR1b Cxcr1 CXCR1 CXCR1 CXCR1 CXCR1a CXCR1bCXCR2 CXCR2 Cxcr2 CXCR2 CXCR2 CXCR2 –CXCR3 CXCR3 Cxcr3 CXCR3 CXCR3 – –CXCR4 CXCR4 Cxcr4 CXCR4 CXCR4 CXCR4 CXCR4CXCR5 CXCR5 Cxcr5 CXCR5 CXCR5 CXCR5 –CXCR6 CXCR6 Cxcr6 CXCR6 CXCR6 CXCR6 CXCR6a CXCR6bCCR1 CCR1 Ccr1 Ccr1l1 CCR1 CCR1L1 CCR1 CCR1 CCR1CCR2 CCR2 Ccr2 CCR2 CCR2 CCR2 CCR2CCR3 CCR3 Ccr3 CCR3 CCR3 CCR3 CCR3CCR4 CCR4 Ccr4 CCR4 CCR4 CCR4 CCR4CCR5 CCR5 Ccr5 CCR5 CCR5 CCR5 CCR5CCR6 CCR6 Ccr6 CCR6 CCR6 CCR6 CCR6CCR7 CCR7 Ccr7 CCR7 CCR7 CCR7 –CCR8 CCR8 Ccr8 CCR8 CCR8 CCR8a CCR8b –CCR9 CCR9 Ccr9 CCR9 CCR9 CCR9 CCR9CCR10 CCR10 Ccr10 CCR10 CCR10 CCR10 –XCR1 XCR1 Xcr1 XCR1 XCR1 XCR1 XCR1CX3CR1 CX3CR1 Cx3cr1 CX3CR1 CX3CR1 CX3CR1 CX3CR1CXCR7 CXCR7 Cxcr7 CXCR7 CXCR7 CXCR7 CXCR7CCBP2 CCBP2 Ccbp2 CCBP2 CCBP2 CCBP2 –CCRL1 CCRL1 Ccrl1 CCRL1 CCRL1 CCRL1 CCRL1CCRL2 CCRL2 Ccrl2 CCRL2 CCRL2 CCRL2 CCRL2DARC DARC Darc DARC DARC DARC –

are shmenc

(

ara2acXo

TC

a The GenBank or Ensembl IDs and amino acid sequences of these receptor genesb Bold letters indicate official gene symbols assigned by the HUGO Gene No

http://www.informatics.jax.org/).

re underway, and they are chosen for the analyses. Mammals andeptiles shared a common ancestor 312 million years ago (Mya),nd birds diverged from reptiles 235 Mya (Benton and Donoghue,007). Amphibians diverged from other tetrapods 330 Mya (Benton

nd Donoghue, 2007). ChemR genes of Xenopus laevis, which islosely related to X. tropicalis, are used when the corresponding. tropicalis genes have not yet been identified. The ChemR genesf these animals are shown in Table 3.

able 3hemokine receptor gene groups of birds, lizard, and froga.

ChemR group Chicken Zebra Finch D

CXCR2CXCR1 & 2 (IL8RB)b – CCXCR3 – – –

CXCR3-like – – –CXCR4 CXCR4 CXCR4 CCXCR5 CXCR5 CXCR5 CCXCR6 – – CCCR1 – – –CCR2 CCR2 CCR2 CCCR3 – – –CCR4 CCR4 CCR4 CCCR5 CCR5 CCR5 CCCR6 CCR6 CCR6 CCCR7 CCR7 CCR7 CCCR8 CCR8a CCR8b CCR8a CCR8b CCR8c CCCR9 CCR9 CCR9 CCCR10 – – –

CCR12 – – –XCR1 XCR1 XCR1 XCX3CR1 CX3CR1 CX3CR1 CCXCR7 CXCR7 CXCR7 CCCBP2 CCBP2 CCBP2a CCBP2b CCCRL1 CCRL1 CCRL1a CCRL1b CCCRL2 – – –DARC – – –

a The GenBank or Ensembl IDs and amino acid sequences of these receptor genes are shb Bold letters indicate official gene symbols assigned by the Chicken Gene Nomenclatuc Xenopus laevis gene.

own in supplementary Table 1 and Fig. 1, respectively.lature Committee (http://www.genenames.org/) or Mouse Genome Informatics

The comparative genomic analyses demonstrate a relativelywell conserved synteny between birds and human (supplementaryFig. 4). While no ChemR genes constituting new ChemR groups areidentified in the birds, ChemRs belonging to the groups of CXCR3,

CCR1, CCR3, CCR10, CCRL2, and DARC are missing in all three birds.The lack of CXCR3, which plays an important role in trafficking ofactivated T cells and Th1 cells in human (Liu et al., 2005), fits withthe absence of its ligands, CXCL4, CXCL4LL1, CXCL9, CXCL10, and

uck Anole Lizard Xenopus

XCR2 CXCR1 CXCR2 cxcr1c

CXCR3 CXCR3CXCR3.2

CXCR3.2 (cxcr3)XCR4 CXCR4 cxcr4 cxcr4-bc

XCR5 CXCR5 CXCR5XCR6 CXCR6 cxcr6

– –CR2 CCR2 –

– –CR4 CCR4 –CR5 CCR5a CCR5b –CR6 CCR6 CCR6CR7 CCR7 ccr7CR8a CCR8b CCR8c – CCR8CR9 CCR9 CCR9

CCR10 CCR10CCR12

– (ccr3)CR1 XCR1 XCR1X3CR1 CX3CR1 –XCR7 CXCR7 cxcr7CBP2 – –CRL1 CCRL1 CCRL1

– –DARC –

own in supplementary Table 1 and Fig. 1, respectively.re Committee (Burt et al., 2009) or Xenbase (http://www.xenbase.org/).

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10 H. Nomiyama et al. / Developmental and

XCL11, in chickens (Kaiser et al., 2005). Given that chickens arenown to lack eosinophils, the birds may not require CCR3 nor itsosinophil-attracting chemokines CCL11, CCL24, and CCL26 (Kaisert al., 2005). Furthermore, since most of the CCR1 and CCR3 ligandsre also recognized by CCR2 and CCR5 in mammals, the lack of CCR1nd CCR3 may be covered by CCR2 and CCR5 in birds. Similarly,CR10, which is critically involved in the accumulation of IgA-ecreting cells to the lactating mammary gland in mice (Morteaut al., 2008), may not be necessary for birds. Accordingly, its ligandsCL27 and CCL28 are also missing in birds (Kaiser et al., 2005). Inhe chicken and duck genomes, there is only one gene that showsimilarity to human CXCR1 and CXCR2 in the genomic region ofynteny. The chicken gene CXCR2 (IL8RB) has been shown to bindwo ligands, K60 and 9E3, both of which are assumed to be therthologs of human CXCL8 (Li et al., 2005; Poh et al., 2008). Welassify the bird gene into the CXCR1 & 2 group (Table 3) becauset is impossible to determine it as CXCR1 or CXCR2. The birds pos-ess two (chicken) or three (duck and zebra finch) CCR8-like genes.CR8a gene is located in a region of conserved synteny betweenuman and the birds. CCR8b gene has been translocated to a regionetween CCR2 and XCR1 genes in the birds, where CCR1 and CCR3enes are located in human. The third gene CCR8c is located adja-ent to the CCR8a gene in duck, while its chromosomal locus is stillnclear in zebra finch.

Previously, three groups have reported genes for chemokinesnd their receptors in the chicken genome (DeVries et al., 2006;aiser et al., 2005; Wang et al., 2005). Although the sequence ver-ions they used are previous ones, their annotations of the ChemRenes are essentially the same as the present one (supplementaryable 2). However, the phylogenetic analyses alone could not cor-ectly determine the orthologous relationships of genes if thearalogous genes underwent gene conversion. Thus, Kaiser et al.2005) called our CCR2, CCR5, and CCR8b as CCRb, CCRa, and CCRc,espectively, while Wang et al. (2005) called our CCR2 and CCR5s CCR5 and CCR2, respectively. Furthermore, when we assign theigands they identified to the receptors, the ligands of all chickeneceptors may have been identified. One exception is CCL25, thenly known ligand of CCR9 in mammals, which has not yet beendentified in chicken.

Like birds, anole lizard appears to lack CCR1 and CCR3. Further-ore, Xenopus lacks CCR2 and CCR5 in addition to CCR1 and CCR3,

ll of which mainly bind inflammatory CC chemokines. Instead,enopus has a new group gene CCR12 (ccr3), which may substi-

ute for the missing receptors. Unlike birds, however, both lizardnd Xenopus possess CXCR3 gene and also a CXCR3-like gene. TheXCR3-like gene is located close to CXCR3 in each genome buthe CXCR3-like genes form a monophyletic group in the phyloge-etic tree. Because zebrafish has a similar CXCR3-like gene termedxcr3.2, the lizard and Xenopus genes are designated CXCR3.2, ande classify these genes into the same orthologous group termedXCR3L (Table 3). When we assign the Xenopus ligands identi-ed by DeVries et al. (2006) or compiled by NCBI Entrez Genehttp://www.ncbi.nlm.nih.gov/gene) to the Xenopus ChemRs, theigands for two receptors, ccr8 and XCR1, may still be missing inhe Xenopus genome.

It appears that Xenopus has only one ‘CXCR1 & 2’ group genexcr1, while anole lizard possesses two, which are named as CXCR1nd CXCR2 based on the synteny conservation. In X. laevis, twoXCR4-like genes, cxcr4-a and cxcr4-b, have been identified, while. tropicalis possesses only one gene cxcr4 corresponding to cxcr4-a.ince X. laevis has arisen from tetraploidization at ∼30 Mya (Bisbee

t al., 1977), X. laevis may duly have two copies of CXCR4 genesompared to nonpolyploid X. tropicalis.

Of the 5 atypical ChemRs found in human, the orthologs ofXCR7, CCBP2 and CCRL1 are present in the birds, while those ofXCR7, CCRL1, and DARC are present in anole lizard and Xenopus.

arative Immunology 35 (2011) 705–715

6. Jawed and jawless fishes

For the fish, we have analysed three ray-finned fishes, zebrafish(Danio rerio, order Cypriniformes), Medaka (Oryzias latipes, orderCyprinodontiformes) and Tetraodon (Tetraodon nigroviridis, orderTetraodontiformes). We have also analysed elephant shark (Cal-lorhinchus milii, order Chimaeriformes, class Chondrichthyes),which is a member of cartilaginous fishes and is the oldest taxonof living jawed vertebrates, and sea lamprey (Petromyzon marinus,order Petromyzontiformes, superclass Agnatha), one of the earli-est jawless vertebrates. Jawed and jawless vertebrates diverged477 Mya (Janvier, 2006), and then jawed vertebrates divided intobony and cartilaginous fishes about 450 Mya (Sansom et al., 1996).The split between bony fishes and ray-finned fishes was about416 Mya (Benton and Donoghue, 2007). The identified ChemRgenes are shown in Table 4. Genes of Takifugu rubripes (orderTetraodontiformes), which is closely related to Tetraodon, are usedwhen the corresponding Tetraodon genes are still missing. Sincethe genomic sequence data of elephant shark and sea lamprey aremostly non-overlapping short scaffolds, it is not still possible tocompare their genome maps with those of other species.

Fish genomes containing ChemR genes are highly rearrangedrelative to those of mammals, and fishes possess often more thantwo genes for a single human counterpart. One reason is the fish-specific whole-genome duplication event, which occurred in ray-finned fishes before the divergence of most teleost species (Meyerand Van de Peer, 2005; Sato and Nishida, 2010). Furthermore, whilethe genomes of Medaka and Tetraodon are well conserved, that ofzebrafish is quite different. Zebrafish is known to have the highestgene duplication rate in the vertebrates (Blomme et al., 2006). Inconsistent with this fact, at least 40 ChemR genes are identifiedin zebrafish, while Medaka and Tetraodon have 31 and 24 ChemRgenes, respectively (Table 4).

From the phylogenetic and genomic analyses, we have ten-tatively classified the fish XCR1-like receptors into three groups,XCR1, XCR1L, and CCR12. The CCR12 group contains zebrafishccr12.1, ccr12.2 and ccr12.3, and the related genes from Medaka,Tetraodon, and Xenopus. The zebrafish XCR1 and XCR1L groupscontain 4 and 3 member genes, respectively. For each group, atleast 3 genes were tandemly duplicated on a single chromosome.Notably, however, the 3 teleosts have only one gene in CXCR3L,CCR7, and CCR10 groups. Like Xenopus, the teleosts have no CCR1,2, 3, and 5 genes. The fish-specific CCR11, XCR1L, or CCR12 groupreceptors may substitute for these missing receptors. The teleostsalso lack CCR4, but contain CCR4 & 8 group receptors, which resem-ble CCR4 and CCR8 of other species.

Previously, we identified over 100 chemokine genes in zebrafishbut only 18 genes in Tetraodon (Nomiyama et al., 2008).We classified zebrafish chemokines into several groups basedon the phylogenetic data. The majority of the genes are fishlineage-specific. The remaining ones are CXCL11, CXCL12, CXCL14,CCL17/20, CCL19/21/25, and CCL27/28. Peatman and Liu, 2007 alsodivided the fish CC chemokines into similar groups. Except forCXCL14, whose receptor is still unknown in mammals, we can pre-dict the receptors for these fish chemokines based on the humanligand-receptor relationships (Fig. 1); CXCL11 (CXCR3 and CXCR7),CXCL12 (CXCR4 and CXCR7), CCL17/20 (CCR6), CCL19/21/25 (CCR7and CCRL1), and CCL27/28 (CCR10). The 20 Tetraodon ChemR genesare also classified into the same groups as those of zebrafish.

Unlike teleost fishes, elephant shark, one of cartilaginous fishes,does not seem to have undergone the fish-specific whole-genome

duplication (Venkatesh et al., 2007). Probably due to this fact,elephant shark possesses in most cases only one gene in eachChemR group. In the elephant shark genome, there exist at least14 genes in 12 ChemR groups. Since the sequence coverage isapproximately 75% (1.4X) (Venkatesh et al., 2007), there may still

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Table 4Chemokine receptor gene groups of jawed and jawless fishesa.

ChemR group Zebrafish Medaka Tetraodon Elephant shark Sea lamprey

CXCR1 & 2 cxcr1b CXCR1c(cxcr2)CXCR3b

CXCR1a CXCR1b CXCR1c CXCR1a CXCR1b CXCR1c CXCR1a CXCR1b –

CXCR3 CXCR3a (cxcr3.1) CXCR3a CXCR3b CXCR3c CXCR3d CXCR3ac CXCR3b – –CXCR3L cxcr3.2 CXCR3.2 CXCR3.2 – –CXCR4 cxcr4a cxcr4b CXCR4c CXCR4d CXCR4a CXCR4b CXCR4a CXCR4b CXCR4 CXCR4CXCR5 cxcr5 CXCR5b CXCR5 CXCR5 CXCR5 –CXCR6 – – – CXCR6 –CCR1 – – – – –CCR2 – – – – –CCR3 – – – – –CCR4 – – – CCR4/8 –CCR4 & 8 CCR4La

(ccr8.1)CCR4Lb(ccr8.2)

CCR4Lc CCR4La CCR4Lb CCR4Lc CCR4La CCR4Lc – –

CCR5 – – – – –CCR6 ccr6a ccr6b CCR6a CCR6b CCR6a CCR6 –CCR7 ccr7 CCR7 CCR7 CCR7 –CCR8 – – – – –CCR9 ccr9a ccr9b CCR9c CCR9a CCR9b CCR9a CCR9b CCR9 –CCR10 ccr10 CCR10 CCR10 CCR10 –CCR11 ccr11.1 ccr11.2 CCR11c

(ccr13)CCR11d CCR11a CCR11b CCR11a – –

CCR12 ccr12.1 ccr12.2 ccr12.3 CCR12.1 CCR12.3 CCR12.1 CCR12.3 –CCR14 – – – – CCR14a CCR14bXCR1 xcr1a XCR1b XCR1c XCR1d XCR1a – XCR1e –XCR1L XCR1La

(xcr1b)XCR1Lc XCR1Ld

(ccr8.3)XCR1La XCR1Lb XCR1Lc XCR1Lac XCR1Lcc XCR1Ld XCR1Le –

CX3CR1 – – – – –CXCR7 cxcr7a cxcr7b CXCR7c CXCR7 CXCR7 CXCR7 CXCR7a CXCR7bCCBP2 – – – – –CCRL1 ccrl1a ccrl1b CCRL1a CCRL1b CCRL1a CCRL1b – –CCRL2 – – – – –DARC – – – – –

a The GenBank or Ensembl IDs and amino acid sequences of these receptor genes are shown in supplementary Table 1 and Fig. 1, respectively.b Bold letters indicate official gene symbols assigned by the ZFIN (http://zfin.org/cgi-bin/webdriver?MIval=aa-ZDB home.apg).c Takifugu rubripes gene.

712 H. Nomiyama et al. / Developmental and Comparative Immunology 35 (2011) 705–715

Mammalia(mammals)

Osteichthyes(bony fish)

Tetrapoda(four-limbedvertebrates)

Amniota(amniotes)

Actinopterygii(ray-finned fish)

Amphibia(amphibians)

Aves(birds)

Reptilia(reptiles)

Vertebrata

CXCR7

CCBP2

CCRL1

CCRL2

DARCDARC

CXCR1 & 2

CXCR3L

CXCR4

CXCR5

CCR6

CCR7

CCR9

CCR11

XCR1

XCR1L

CXCR7

CCRL1

CXCR1 & 2

CXCR3CXCR3

CXCR3L

CXCR4

CXCR5

CCR6

CCR7

CCR9

XCR1XCR1

CXCR7

CCRL1

CXCR1 & 2

CXCR3

CXCR3L

CXCR4

CXCR5

CXCR6CXCR6CXCR6

CCR2

CCR4

CCR5

CCR6

CCR7

CCR9

CCR10CCR10CCR10CCR10

XCR1

CCR12 CCR12CCR12

CX3CR1

CXCR7

CCRL1

CXCR1 & 2

CXCR4

CXCR5

CXCR6

CCR2

CCR4

CCR5

CCR6

CCR7

CCR8CCR8

CCR9

XCR1

CX3CR1

CXCR7

CCBP2

CCRL1

Gnathostomata(jawed

vertebrates)

Agnatha(jawless fish)

Cephalochordata(amphioxus)

CXCR4

CXCR7

CCR14

Teleostomi(bony vertebrates

with jaws)

Chondrichthyes(cartilaginous

fish)

CXCR1 & 2

CXCR4

CXCR5

CCR4

CCR6

CCR7

CCR9

CXCR7

CXCR1

CXCR2

CXCR3

CXCR4

CXCR5

CXCR6

CCR1

CCR2

CCR3

CCR4

CCR5

CCR6

CCR7

CCR9

CCR10

XCR1

CX3CR1

CCR8

CCR4 & 8

111 3 1 3 61Number of species analyzed

1R 2R

3R

inflammatory homeostatic dual-function atypical

550 Mya 477 Mya 450 Mya 416 Mya 330 Mya 312 Mya

235 Mya

*****

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ig. 2. Evolutionary history of the chemokine receptor orthologous groups. The diverased on fossil records (Benton and Donoghue, 2007; Janvier, 2006; Sansom et al., 1nd the fish-specific genome duplication (3R) are also shown. The mammalian Chcartilaginous fish) CCR4/8 gene is provisionally included in the CCR4 orthologous g

e other ChemR genes in the elephant shark genome. Interestingly,he shark genome contains CXCR6 and CCR4-like genes, which theeleost fishes lack, suggesting their loss during the evolution ofctinopterygians. The grouping of elephant shark CCR4-like, how-

ver, cannot be specified because it clusters with CCR8s or CCR4s ofther species depending on the phylogenetic tree used. It is there-ore provisionally designated as CCR4/8.

Since we could not identify any potential ChemRs in theenomes of amphioxus (Branchiostoma floridae) and sea squirts

times (Mya, million years ago) shown are the minimum divergence times estimatedhe timings of the two successive rounds of whole-genome duplication (1R and 2R)

present in the major gene cluster are indicated with asterisks. The elephant shark

(Ciona intestinalis), the genome of the jawless fish lamprey offersthe best possibility of finding the primitive ChemR genes. In addi-tion to the CXCR4 gene (Kuroda et al., 2003), there exist two pairs ofChemR genes, Cxcr 1 and Cxcr 2, and Ccr 1 and Ccr 2 (Bajoghli et al.,

2009) in the lamprey. Cxcr 1 and Cxcr 2 resemble CXCR7, whileCcr 1 and Ccr 2 constitute a new group CCR14. CXCR4 and CXCR7are known to play important roles in the mammalian and teleostdevelopment by binding CXCL12 as the signaling receptor and thescavenging receptor, respectively (Thelen and Thelen, 2008). Thus,

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H. Nomiyama et al. / Developmental and

he same set of ChemRs and CXCL12 may play a similar develop-ental role since the first appearance of the chemokine system in

he lamprey.

. Evolutionary history of the chemokine receptor genes

Fig. 2 summarizes the gain and loss events of ChemR genes dur-ng the vertebrate evolution. Vertebrates are a group of chordatenimals that contains the urochordates (sea squirts) and cephalo-hordates (amphioxus). Since we and others (Bajoghli et al., 2009,eVries et al., 2006) have failed to identify ChemRs in the genomesf amphioxus and sea squirts, agnathan lamprey is so far the mostrimitive vertebrate that possesses the ChemRs. In the lamprey,hree CXCL genes (CXCL8-like, CXCL15-like and CXCL12) and twoCL chemokine genes have been identified (Bajoghli et al., 2009;omiyama et al., 2010). The two CCL chemokines may bind to theCR14 group receptors, while CXCL12 is likely to bind to CXCR4 andhe CXCR7 group (Cxcr 1 and Cxcr 2). Given that the receptor(s) forXCL8-like and CXCL15-like chemokines are yet to be discovered,o the total of at least 5 ChemR groups may exist in the lamprey.he emergence of chemokine system in the lamprey coincides withhe appearance of lymphocytes and adaptive immune responses ingnathans, although the immune system of agnathans is quite dif-erent from that of cartilaginous fishes (Cooper and Alder, 2006).uising et al. (2003a) have proposed that the original biological

ole of the CXC chemokines and their receptors might be in theentral nervous system before they were adapted by the immuneystem. However, this hypothesis may be disputable (Huising et al.,003b; Shields, 2003). Given that the vertebrate nervous systemriginates from the basal chordate amphioxus (Holland, 2009) andhe chemokines and their receptors are not yet found in amphioxus,he original role of the chemokine system might have been ratherelated to the immune system in the ancient jawless vertebrates. Its noteworthy that the lamprey CXCR4 is expressed in lymphocyte-ike cells (Kuroda et al., 2003). It is therefore of interest to know theypes of cells expressing CCR14 and their biological role in lamprey.

The entire genome is assumed to have been duplicated in twoounds at the dawn of vertebrate evolution (Meyer and Van de Peer,005; Sato and Nishida, 2010). One round of whole-genome dupli-ation (1R) occurred before the divergence of jawless and jawedertebrates, and the second one (2R) after the divergence of theawless and jawed vertebrate lineages but before the split of carti-aginous fish and bony fish lineages (Venkatesh et al., 2007) (Fig. 2).n additional event (3R) leading to at least up to eight copies of

he ancestral genome might have occurred in the stem lineage ofay-finned fishes after their divergence from the land vertebratesMeyer and Van de Peer, 2005; Sato and Nishida, 2010). The pres-nce of homeobox (Hox) gene clusters on four chromosomes inost vertebrates but only on one chromosome in invertebrates

as been taken as the evidence for two-rounds of whole-genomeuplication (Lundin et al., 2003). Since two or more Hox-bearinghromosomes contain paralogs of a large number of gene fami-ies, these genes were assumed to be duplicated together with theox clusters by polyploidization. DeVries et al. (2006) proposedmodel in which the ChemR genes increased their numbers by

uplications concomitant with the Hox gene clusters. However,ontroversy still exists regarding the extent of the duplicated chro-osome segments (Abbasi and Grzeschik, 2007; Hughes et al.,

001; Larhammar et al., 2002). The controversy may largely stemrom too much dependence on the phylogenetic data due to the

ncomplete assemblies of the various animal genomes. Compar-son of the genome maps of the regions spanning both the Hoxlusters and ChemR genes, especially those of agnathans and car-ilaginous fishes, may elucidate whether the ChemR genes wereuplicated together with the Hox clusters by tetraploidization or

arative Immunology 35 (2011) 705–715 713

translocated to some of the Hox-bearing chromosomes duringevolution.

The cartilaginous fish elephant shark and the tetrapods containfour Hox clusters, whereas the lamprey contains at least three Hoxclusters, one of which seems to be the result of a lineage-specificduplication event (Force et al., 2002). Compared to the lamprey,which possesses 5 genes (3 ChemR groups), elephant shark con-tain 14 genes (12 ChemR groups). The presence of four times thenumber of ChemR groups in elephant shark as in lamprey suggeststhat ChemR duplication events might have occurred in the cartilagi-nous fish lineage besides the second whole-genome duplication.Furthermore, 9 of the 12 elephant shark ChemR groups are amongthe 11 homeostatic or dual function receptors in mammals. Amongthe rest of the three receptors (CXCR1 & 2, CCR12, and CXCR7),only CCR12 is not present in the mammals. These results suggestthat the basic components of the ChemRs, in particular the home-ostatic and dual function classes, had been well established beforethe divergence of cartilaginous and bony fishes.

Compared to elephant shark, the teleost fishes contain moreChemR genes and groups. Although two ChemR groups (CXCR6and CCR4) were lost in the teleosts, 6 novel groups were gener-ated, making the total number of the ChemR groups to 16. Amongthe newly emerged ChemR groups are CXCR3, CXCR3L, and CCRL1,all of which have been transmitted to mammals and/or birds. Theother 3 groups (CCR4 & 8, CCR11, and XCR1L) were teleost-specificand lost in the other lineages. There are total 24 and 31 ChemRgenes in Tetraodon and Medaka, respectively. On the other hand,there are 41 genes in zebrafish. This may be in part due to theadditional fish-specific whole-genome duplication, but local dupli-cation events may also have contributed to the increases. Since theappearance of tetrapods, new ChemR groups (CCR1, CCR2, CCR3,CCR5, CCR8, CX3CR1, CCBP2, CCRL2 and DARC) have emerged.Among these, CCR1, CCR3, and CCRL2 were generated recently inthe mammalian lineage. There are also gene losses in the bird lin-eage. Similarly, some of the tetrapod-specific group genes mighthave been deleted in the lower vertebrates. For example, DARC ishighly divergent from other ChemRs probably due to its ancient ori-gin but only present in higher vertebrates. Eventually, mammalscontain 23 groups (23 genes in human) compared to 12 groups(14 genes) in elephant shark. Since no whole-genome duplicationevent has occurred during the evolution from cartilaginous fishesto mammals, recurrent local gene duplications may have amplifiedthe diversity of the ChemR system in mammals.

As described above, most extant homeostatic and dual functionChemRs were generated at the early stages of vertebrate evo-lution. In contrast, most of the ChemR genes supposed to haveinflammatory functions might not have been transmitted to highervertebrates during the vertebrate evolution so that the mammalianinflammatory ChemRs have originated in amniotes. Such short life-spans of inflammatory ChemR genes might be in part due to thesurvival strategy used by host animals to avoid the viral mimicry ofchemokines and their receptors (Murphy, 2001). One exception isthe CXCR1 & 2 group genes, which had originated in jawless or car-tilaginous fish and have been retained throughout the vertebrateevolution. From these considerations, it may not be necessarily truethat the ancestor ChemR is a homeostatic one. It is therefore inter-esting to see which class the lamprey CCR14 group genes belong toand what class of ChemR is a prototype.

8. Conclusion

The application of human nomenclature system to lower ani-mals has caused a substantial confusion in the naming of vertebrateChemR genes. We have therefore comprehensively surveyed theChemR genes of 16 vertebrate species and classified the genes

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14 H. Nomiyama et al. / Developmental and

hrough the combination of phylogenetic and comparative genomicnalyses. The results show that there are 29 ChemR orthologousroups in vertebrates and that the gain and loss of the groupsccur as a continuous process in all lineages even after the whole-enome duplication events. Our orthologous grouping may providehe basis for a unified nomenclature of ChemR genes and maylso be useful to understand the function of each ChemR groupembers. A rough evolutionary history of the ChemR genes is

lso obtained. The ChemR genes first emerged in the genome ofgnathan, the most primitive vertebrate, and the CXCR4/CXCR7-XCL12 axis might have be already operative in this lineage. ThehemR genes then expanded and the fundamental types of ChemRsad been established in cartilaginous fishes and further increased

n the diversity in teleosts and higher vertebrates. The complexegulatory requirement of the immune system in higher verte-rates may have accelerated the expansion and diversification ofhe ChemR gene family.

cknowledgements

The original sequencing data of the following species haveeen provided freely by the Beijing Genomics Institute (duck),he Broad Institute (opossum, anole lizard), the Department ofnergy’s Joint Genome Institute (X. tropicalis, Fugu), the Genomeequencing Center at Washington University School of Medicine int. Louis (platypus, chicken, zebra finch, lamprey), and GenoscopeTetraodon) for use in this publication.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.dci.2011.01.019.

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