熊本大学学術リポジトリ. Kumamoto University Repositor

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1 熊本大学学術リポジトリ Kumamoto University Repositor Title A family tree of vertebrate chemoki a unified nomenclature Author(s) Nomiyama, Hisayuki; Osada, Naoki; Y CitationDevelopmental and Comparative Immun Issue date Type URL Right Article Elsevier Ltd All rights rese

2 Ms. Ref. No.: DCI-D Review A family tree of vertebrate chemokine receptors for a unified nomenclature Hisayuki Nomiyama a,*, Naoki Osada b, Osamu Yoshie c a Department of Molecular Enzymology, Kumamoto University Faculty of Life Sciences, Honjo, Kumamoto , Japan b Division of Evolutionary Genetics, Department of Population Genetics, National Institute of Genetics, Mishima, Shizuoka , Japan c Department of Microbiology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka , Japan * Corresponding author. Tel.: ; fax: address: nomiyama@gpo.kumamoto-u.ac.jp Keywords: Chemokine receptors, Gene Cluster, Evolution, Gene duplication, Vertebrates Contents 1. Introduction 2. Sequence and genomic data of vertebrate chemokine receptor genes 3. Nomenclature 4. Mammals 4.1 Human and mouse 4.2 Other mammals 5. Birds, lizard, and frog 6. Jawed and jawless fishes 7. Evolutionary history of the chemokine receptor genes 8. Conclusion

3 ABSTRUCT Chemokines receptors are involved in the recruitment of various cell types in inflammatory and physiological conditions. There are 23 known chemokine receptor genes in the human genome. However, it is still unclear how many chemokine receptors exist in the genomes of various vertebrate species other than human and mouse. Moreover, the orthologous relationships are often obscure between the genes of higher and lower vertebrates. In order to provide a basis for a unified nomenclature system of the vertebrate chemokine receptor gene family, we have analyzed the chemokine receptor genes from the genomes of 16 vertebrate species, and classify them into 29 orthologous groups using phylogenetic and comparative genomic analyses. The results reveal a continuous gene birth and death process during the vertebrate evolution and an interesting evolutionary history of the chemokine receptor genes after the emergence in agnathans. 1. Introduction Chemokines are a multigene family of small, secreted cytokines mediating cell migration during inflammation, immune surveillance, and organogenesis (Moser et al., 2004, Zlotnik and Yoshie, 2000). Chemokines are subdivided into 5 subfamilies (CXC, CC, XC, CX3C, and CX) based on the arrangement of four conserved cysteine residues involved in the formation of disulfide bonds (Nomiyama et al., 2010). In the CXC and CX3C chemokine subfamily, one or three amino acids are inserted between the first two of the four cysteine residues. In the CC subfamily, the first two cysteines are arranged in adjacent positions. The first and third cysteine residues are absent in the XC (or C) subfamily with only one disulfide bond. Furthermore, we have recently described yet another subfamily CX from the analysis of Zebrafish chemokine genes (Nomiyama et al., 2008). The CX subfamily lacks one of the first two cysteine residues but not the third one. Besides the structural criteria, chemokines can be categorized into several groups from the functional point of view (Mantovani et al., 2006, Nomiyama et al., 2010). Inflammatory chemokines are those upregulated in inflammatory conditions and involved in the robust recruitment of leukocytes to inflamed sites. Homeostatic chemokines are those produced constitutively at non-inflamed sites and involved in homeostatic migration and homing of cells in physiological conditions. Some chemokines have both properties, and are thus called dual-function chemokines. In addition, some chemokines are constitutively expressed at high levels and are present in 2

4 the blood stream as inactive forms (Berahovich et al., 2005, Mortier et al., 2008) or stored in alpha-granules of platelets (Brandt et al., 2000). The plasma chemokines can be activated by proteolytic cleavage at inflamed sites, while platelet chemokines are released upon platelet activation. Thus, they may be considered as a kind of inflammatory chemokines. To date, chemokines have only been described in vertebrates (Bajoghli et al., 2009, DeVries et al., 2006). The human genome contains more than 44 members (Zlotnik et al., 2006). The chemokine genes have been evolved rapidly, both in their sequences and the size of gene family (Nomiyama et al., 2010). Thus, the orthologous relationships of some chemokines between the species could be unclear. Especially the chemokines between fishes and other vertebrates are highly divergent (Nomiyama et al., 2008). All the known chemokine receptors (ChemRs) are seven transmembrane domain G protein-coupled receptors (GPCRs) that commonly couple to the G i class of the heterotrimeric G proteins. ChemRs are classified according to the subfamily of chemokine ligands that they bind; CXCR, CCR, XCR, and CX3CR. It is still unknown whether there is a specific group of receptors for CX chemokines in zebrafish (Nomiyama et al., 2008). So far, 18 genes encoding ChemRs with standard chemotactic functions have been identified in the human genome; 6 CXCR genes, 10 CCR genes, 1 XCR gene, and 1 CX3CR gene. ChemRs often bind more than one chemokine, while a single chemokine often binds to more than one receptor. This binding promiscuity is one of the characteristic features of the chemokine system and is mostly observed between inflammatory and plasma chemokines and their receptors (Mantovani, 1999). In contrast, homeostatic chemokines and their receptors display more specific relationships (Yoshie et al., 1997, 2001). Furthermore, 5 genes encoding atypical (non-signaling) ChemRs have been identified (DARC, CCBP2, CCRL1, CCRL2, CXCR7) (Graham, 2009, Leick et al., 2010, Naumann et al., 2010). These atypical ChemRs are often called 'silent' or 'decoy' receptors since they bind chemokines but do not elicit standard chemotactic responses following ligand binding. They are considered to be the scavengers for excess chemokines. Although the functional property of CXCR7 is still controversial (Thelen and Thelen, 2008), it has been shown to act as a scavenger receptor for CXCL12 and CXCL11 (Naumann et al., 2010). Compared to the chemokine ligand genes, the ChemR genes are relatively well conserved across the vertebrate species. However, because of a fish-specific 3

5 whole-genome duplication event, which occurred in the stem lineage of ray-finned (actinopterygian) fishes (including teleosts) after divergence from the land vertebrates (Meyer and Van de Peer, 2005, Sato and Nishida, 2010), together with their higher rates of gene rearrangement and faster evolution of protein sequences compared to mammals, there occurred large increases in the number of genes in the teleost fishes (Ravi and Venkatesh, 2008). This makes it quite difficult to assign the orthologous relationships for the members of multigene families between teleost fishes and other vertebrates. Nevertheless, it may be still desirable to have a unified nomenclature system that covers all the vertebrate ChemR genes. Several groups have identified ChemR genes from various vertebrate species using comparative genomics-based data mining (DeVries et al., 2006, Kaiser et al., 2005, Liu et al., 2009, Wang et al., 2005). However, the draft genome sequences they used were the old versions, and the numbers of species investigated were limited. To provide a more up-to-date view of the ChemR genes of vertebrates, we have made a census of the vertebrate ChemR genes using the latest versions of 16 representative vertebrate genomes 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 combination of phylogenetic and synteny-based approaches using several species is quite useful to resolve the evolutionary history of a gene family (Nomiyama et al., 2010). In the present study, therefore, we have chosen only those animals, whose genome-wide sequencing is in progress or has already been finished, and performed robust and through phylogenetic and genomic analyses. If available, we have also used multiple species of the same animal group but not belonging to the same order so that the obtained ChemR gene content of each species complements each other and the combined data represent the ChemR entity of the animal group. From such analyses, a plausible evolutionary history of ChemR genes could be drawn. Indeed, the obtained results show that continuous gene expansion and contraction events occurred at different time points during the vertebrate evolution. We have also classified the vertebrate ChemR genes into 29 orthologous groups, each of which contains 0 to 4 paralogous genes from one species. 2. Sequence and genomic data of vertebrate chemokine receptor genes We have identified most of the vertebrate ChemR genes in the NCBI or Ensembl annotation. Supplementary Table 1 lists the IDs of their amino acid sequences. We have 4

6 also identified some ChemR genes by BLAST search. There is, however, a possibility that the genes predicted without cdna sequences may include pseudogenes. In supplementary Fig. 1, we show the full amino acid sequence of each gene with DRY (Rovati et al., 2007), CWLP (Shi et al., 2002), TXP (Govaerts et al., 2001), and NPxxY(x) 5,6 F (Fritze et al., 2003) motifs highlighted. The versions of the assembled genome sequences used are also shown in the figure. In supplementary Fig. 2, we show the phylogenetic trees that were constructed using the neighbor-joining method with Dayhoff's (PAM) matrix and removing gaps by pairwise deletion (Saitou and Nei, 1987) (supplementary Fig. 2). We used the sequences corresponding to the ChemR domain pfam00001 (Finn et al., 2010) in the tree construction to exclude the relatively non-conserved N- and C-terminal sequences (supplementary Fig. 3). The tree shown in (B) of supplementary Fig. 2 is constructed by excluding 4 incomplete sequences, platypus CXCR6a and CXCR6b, Tetraodon CCR11a, and elephant shark CXCR4, since adding incomplete sequences in the tree construction may obscure the orthology (Shields, 2003). In supplementary Fig. 4, we show the comparative genomic maps drawn by using Ensembl data. 3. Nomenclature Genes of several vertebrate species, human, mouse, chicken, Xenopus, and zebrafish, have their own 'official' gene symbols assigned by the respective nomenclature committees. As for the human ChemRs, a systematic nomenclature was proposed (Murphy, 2002, Murphy et al., 2000), and the nomenclature committee for human genome organization adopted this system, in which each receptor is identified by its ligand subfamily plus R (receptor) and a given identifying number. For example, CXCR1 refers to CXC chemokine receptor 1, whose representative common name is IL8R. In this review, we propose to categorize vertebrate ChemR genes into 29 orthologous groups based on both the phylogenetic trees and genomic synteny. The orthologous groups are basically defined by the human ChemR members, and thus the name of each group is based on the representative human gene. Each orthologous group includes a true ortholog having evolved from a common ancestor and their paralogs having arisen from speciesor lineage-specific duplication events. In the case of groups without human genes, we have given new names: CCR11, CCR12, and CCR14. Since CCR13 is already used for a zebrafish gene, CCR13 is not used for a group name. 5

7 The gene symbols of the species other than human are in general given the same names as their human orthologs. However, when there are two or more paralogous genes resembling a single human gene, there is no unified rule to resolve such cases. For example, zebrafish gene symbols ccr6a and ccr6b are assigned to genes resembling human CCR6, while symbols ccr8.1 and ccr8.2 are allocated to two paralogous genes resembling human CCR8. In this review, we adhere to the gene symbols assigned by the committees where appropriate, and alphabets in small letters are added to distinguish the genes in the same group, for example, CXCR1a and CXCR1b. In some cases, genes from different species with the same gene symbol are not orthologous. For example, Xenopus ccr3 gene was classified into a newly defined CCR12 group by our analyses, and the gene is thus named as CCR12 (ccr3) to show that the gene having the gene symbol ccr3 belongs to the group CCR12. The names of the ChemR genes reported in the various papers and the names used in this review are summarized in supplementary Table Mammals 4.1 Human and mouse There are 23 known ChemR genes in the human genome, while one additional gene termed Ccr1l1 (Ccr1-like 1) exists in the mouse genome (Table 1). Fig. 1 shows the genomic organization of the human ChemRs and their chemokine ligands. The signaling receptors for CXCL14, CXCL17, and CCL18 are as yet unknown. There is one major gene cluster of ChemRs on the human chromosome 3. The cluster spans a large region of approximately 13 Mb in length containing 12 genes encoding for CXCR6, CCR1 to 5, 8 and 9, XCR1, CX3CR1, and two atypical receptors CCBP2 and CCRL2. Another atypical receptor gene CCRL1 resides 86 Mb apart from the major cluster on the same human chromosome. Although most of the receptors in the cluster interact with the inflammatory chemokines, the receptors CCR9, CXCR6, and XCR1 bind homeostatic or dual-function chemokines and their genes are also arranged next to each other in the middle of the cluster. Phylogenetically, CCR9 and CXCR6 are closely related but are relatively distantly related from the other ChemRs in the cluster (supplementary Fig. 2). The CX3CR1 gene resides between the genes for CCR4 and CCR8, and they are also closely clustered in the tree (supplementary Fig. 2). The remaining ChemR genes are found as individual genes or in mini-clusters (Fig. 1). 6

8 Overall, the genomic organization of mouse ChemR genes is quite similar to that of the human genes (supplementary Fig. 4). However, mouse atypical ChemR genes Ccrl1 (Ccr-like-1) and Ccrl2 are organized differently from the human counterparts. While human CCRL2 gene is located at one end of the major cluster and CCRL1 is located apart from the CCRL2 gene, both Ccrl1 and Ccrl2 genes in the mouse genome, which are closely linked, are translocated to the opposite end of the cluster. Moreover, mouse Ccr1l1, which is not present in the human genome, is located between Ccr1 and Ccr3 in the major cluster. Ccr1l1 is closely related to Ccr1 and grouped in the CCR1 orthologous group together with Ccr1 (Table 2). Although the Ccr1l1 is not characterized in detail (Gao and Murphy, 1995, Nibbs et al., 1997, Perelygin et al., 2008), it contains the DRY-like motif for the G protein coupling as well as the other ChemR motifs (supplementary Fig. 1), suggesting its signaling capacity. Like chemokine ligands, ambiguous orthologous relationships are present for some ChemR genes in the clusters. Paralogous CXCR1 and CXCR2 genes in the CXCR mini-cluster have been shown to undergo gene conversion in the human and rabbit genomes (Shields, 2000). Evidence for gene conversions between CCR2 and CCR5 and between CCR1 and CCR3 has also been shown in a number of mammals (Carmo et al., 2006, Esteves et al., 2007, Perelygin et al., 2008, Shields, 2000, Vazquez-Salat et al., 2007). Since gene conversion homogenizes the nucleotide sequences of paralogous genes, the orthologous relationships could not be defined only by phylogenetic analysis (Shields, 2003). Like chemokine ligands, ChemRs are known to form homo- or hetero-dimers (Thelen et al., 2010). Such dimer formation may modulate the receptor activity, although the physiological relevance of the receptor oligomerization still largely remains unclear. However, it is plausible that gene conversion between adjacent genes such as CCR1 and CCR3 would promote not only their sharing 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 analyzed four mammalian species: cow (Bos taurus, order Artiodactyla), dog (Canis familiaris, order Carnivora), opossum (Monodelphis domestica, order Didelphimorphia, Infraclass Metatheria), and platypus (Ornithorhynchus anatinus, order Monotremata, subclass Prototheria). In contrast to the 7

9 chemokine genes (Nomiyama et al., 2010), one-to-one orthologous relationships are mostly evident for the ChemR genes between human and other mammals, and a highly conserved synteny is maintained across the mammalian species investigated (supplementary Fig. 4). In the cow and dog genomes, however, the major gene cluster is split into two. While the two regions are located apart from each other on the same chromosome in the cow genome, they reside on separate chromosomes in the dog genome. As seen in the mouse genome, the atypical ChemRs CCRL1 and CCRL2 are closely located in the opossum and platypus major clusters, strengthening that it was originally generated by a local duplication in the major cluster. Furthermore, some ChemR genes may be still missing in the opossum and platypus genomes. For example, both opossum and platypus appear to lack CXCR3, but two (CXCL9 and CXCL10) and one (CXCL10) of the CXCR3 ligand genes have been identified in the opossum and platypus genomes, respectively (Nomiyama et al., 2010). Similarly, the CXCR5 gene has not been found in platypus, but its ligand CXCL13 gene exists in the genome (Nomiyama et al., 2010). Several genes were duplicated in some species. CCR1L1 is found between CCR1 and CCR3 genes in the cow genome as in the mouse genome. The genes for CCR8 and CXCR6 were duplicated in the opossum and platypus genomes, respectively. The platypus genome contains two genes similar to human CXCR1 and CXCR2. It is however impossible to determine which gene is orthologous to human CXCR1 or CXCR2 from the genomic comparison due to the lack of markers around both genes. The platypus genes are therefore tentatively designated CXCR1a and CXCR1b. 5. Birds, lizard, and frog Three species were selected from the birds for the analyses: chicken (Gallus gallus, order Galliformes), zebra finch (Taeniopygia guttata, order Passeriformes), and duck (Anas platyrhynchos, order Anseriformes). Out of reptiles and amphibians, the genome sequencing of the anole lizard (Anolis carolinensis, order Squamata, class Reptilia) and Xenopus tropicalis (order Anula, class Amphibia) are underway, and they are chosen for the analyses. Mammals and reptiles shared a common ancestor 312 million years ago (Mya), and birds diverged from reptiles 235 Mya (Benton and Donoghue, 2007). Amphibians diverged from other tetrapods 330 Mya (Benton and Donoghue, 2007). ChemR genes of Xenopus laevis, which is closely related to Xenopus tropicalis, are used when the corresponding Xenopus tropicalis genes have not yet been identified. The 8

10 ChemR genes of these animals are shown in Table 3. The comparative genomic analyses demonstrate a relatively well conserved synteny between birds and human (supplementary Fig. 4). While no ChemR genes constituting new ChemR groups are identified 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 of activated T cells and Th1 cells in human (Liu et al., 2005), fits with the absence of its ligands, CXCL4, CXCL4LL1, CXCL9, CXCL10, and CXCL11, in chickens (Kaiser et al., 2005). Given that chickens are known to lack eosinophils, the birds may not require CCR3 and its eosinophil-attracting chemokines CCL11, CCL24, and CCL26 (Kaiser et al., 2005). Furthermore, since most of the CCR1 and CCR3 ligands are also recognized by CCR2 and CCR5 in mammals, the lack of CCR1 and CCR3 may be covered by CCR2 and CCR5 in birds. Similarly, CCR10, which is critically involved in the accumulation of IgA-secreting cells to the lactating mammary gland in mice (Morteau et al., 2008), may not be necessary for birds. Accordingly, its ligands CCL27 and CCL28 are also missing in birds (Kaiser et al., 2005). In the chicken and duck genomes, there is only one gene that shows similarity to human CXCR1 and CXCR2 in the genomic region of synteny. The chicken gene CXCR2 (IL8RB) has been shown to bind two ligands, K60 and 9E3, both of which are assumed to be the orthologs of human CXCL8 (Li et al., 2005, Poh et al., 2008). We classify the bird gene into the CXCR1 & 2 group (Table 3) because it is impossible to determine it as CXCR1 or CXCR2. The birds possess two (chicken) or three (duck and zebra finch) CCR8-like genes. CCR8a gene is located in a region of conserved synteny between human and the birds. CCR8b gene has been translocated to a region between CCR2 and XCR1 genes in the birds, where CCR1 and CCR3 genes are located in human. The third gene CCR8c is located adjacent to the CCR8a gene in duck, while its chromosomal locus is still unclear in zebra finch. Previously, three groups have reported genes for chemokines and their receptors in the chicken genome (DeVries et al., 2006, Kaiser et al., 2005, Wang et al., 2005). Although the sequence versions they used are previous ones, their annotations of the ChemR genes are essentially the same as the present one (supplementary Table 2). However, the phylogenetic analyses alone could not correctly determine the orthologous relationships of genes if the paralogous genes underwent gene conversion. Thus, Kaiser et al. (2005) called our CCR2, CCR5, and CCR8b as CCRb, CCRa, and CCRc, 9

11 respectively, while Wang et al. (2005) called our CCR2 and CCR5 as CCR5 and CCR2, respectively. Furthermore, when assigning the ligands they identified to the receptors, the ligands of all chicken receptors may have been identified. One exception is CCL25, the only known ligand of CCR9 in mammals, which has not yet been identified in chicken. Like birds, anole lizard appears to lack CCR1 and CCR3. Furthermore, Xenopus lacks CCR2 and CCR5 in addition to CCR1 and CCR3, all of which mainly bind inflammatory CC chemokines. Instead, Xenopus has a new group gene CCR12 (ccr3), which may substitute for the missing receptors. Unlike birds, however, both lizard and Xenopus possess CXCR3 gene and also a CXCR3-like gene. The CXCR3-like gene is located close to CXCR3 in each genome but the CXCR3-like genes form a monophyletic group in the phylogenetic tree. Because zebrafish has a similar CXCR3-like gene termed cxcr3.2, the lizard and Xenopus genes are designated CXCR3.2, and we classify these genes into the same orthologous group termed CXCR3L (Table3). When we assign the Xenopus ligands identified by DeVries et al. (2006) or compiled by NCBI Entrez Gene ( ) to the Xenopus ChemRs, the ligands for two receptors, ccr8 and XCR1, may still be missing in the Xenopus genome. It appears that Xenopus has only one 'CXCR1 & 2' group gene cxcr1, while anole lizard possesses two, which are named as CXCR1 and CXCR2 based on the synteny conservation. In X. laevis, two CXCR4-like genes, cxcr4-a and cxcr4-b, have been identified, while X. tropicalis possesses only one gene cxcr4 corresponding to cxcr4-a. Since X. laevis has arisen from tetraploidization at ~30 Mya (Bisbee et al., 1977), X. laevis may duly have two copies of CXCR4 genes compared to nonpolyploid X. tropicalis. Of the 5 atypical ChemRs found in human, the orthologs of CXCR7, CCBP2 and CCRL1 are present in the birds, while those of CXCR7, CCRL1, and DARC are present in anole lizard and Xenopus. 6. Jawed and jawless fishes For the fish, we have analyzed three ray-finned fishes, zebrafish (Danio rerio, order Cypriniformes), Medaka (Oryzias latipes, order Cyprinodontiformes) and Tetraodon (Tetraodon nigroviridis, order Tetraodontiformes). We also analyzed elephant shark (Callorhinchus milii, order Chimaeriformes, class Chondrichthyes), which is a member of cartilaginous fishes and is the oldest taxon of living jawed vertebrates, and sea lamprey 10

12 (Petromyzon marinus, order Petromyzontiformes, superclass Agnatha), one of the earliest jawless vertebrates. Jawed and jawless vertebrates diverged 477 Mya (Janvier, 2006), and then jawed vertebrates divided into bony and cartilaginous fishes about 450 Mya (Sansom et al., 1996). The split between bony fishes and ray-finned fishes was about 416 Mya (Benton and Donoghue, 2007). The identified ChemR genes are shown in Table 4. Genes of Takifugu rubripes (order Tetraodontiformes), which is closely related to Tetraodon, are used when the corresponding Tetraodon genes are still missing. Since the genomic sequence data of elephant shark and sea lamprey are mostly non-overlapping short scaffolds, it s not still possible to compare their genome maps with those of other species. Fish genomes containing ChemR genes are highly rearranged relative to those of mammals, and fishes possess often more than two 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 (Meyer and Van de Peer, 2005, Sato and Nishida, 2010). Furthermore, while the genomes of Medaka and Tetraodon are well conserved, that of zebrafish is quite different. Zebrafish is known to have the highest gene duplication rate in the vertebrates (Blomme et al., 2006). In consistent with this fact, at least 40 ChemR genes are identified in zebrafish, while Medaka and Tetraodon have 31 and 24 ChemR genes, respectively (Table 4). From the phylogenetic and genomic analyses, we have tentatively classified the fish XCR1-like receptors into three groups, XCR1, XCR1L, and CCR12. The CCR12 group contains zebrafish ccr12.1, ccr12.2 and ccr12.3, and the related genes from Medaka, Tetraodon, and Xenopus. The zebrafish XCR1 and XCR1L groups contain 4 and 3 member genes, respectively. For each group, at least 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 group receptors may substitute for these missing receptors. The teleosts also lack CCR4, but contain CCR4 & 8 group receptors, which resemble CCR4 and CCR8 of other species. Previously, we identified over100 chemokine genes in zebrafish but only 18 genes in Tetraodon (Nomiyama et al., 2008). We classified zebrafish chemokines into several groups based on the phylogenetic data. The majority of the genes are fish lineage-specific. The remaining ones are CXCL11, CXCL12, CXCL14, CCL17/20, 11

13 CCL19/21/25, and CCL27/28. Peatman et al. (2007) also divided the fish CC chemokines into similar groups. Except for CXCL14, whose receptor is still unknown in mammals, we can predict the receptors for these fish chemokines based on the human ligand-receptor relationships (Fig. 1); CXCL11 (CXCR3 and CXCR7), CXCL12 (CXCR4 and CXCR7), CCL17/20 (CCR6), CCL19/21/25 (CCR7 and CCRL1), and CCL27/28 (CCR10). The 20 Tetraodon ChemR genes are 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 each ChemR group. In the elephant shark genome, there exist at least 14 genes in 12 ChemR groups. Since the sequence coverage is approximately 75% (1.4X) (Venkatesh et al., 2007), there may still be other ChemR genes in the elephant shark genome. Interestingly, the shark genome contains CXCR6 and CCR4-like genes, which the teleost fishes lack, suggesting their loss during the evolution of actinopterygians. The grouping of elephant shark CCR4-like, however, cannot be specified because it clusters with CCR8s or CCR4s of other species depending on the phylogenetic tree used. It is therefore provisionally designated as CCR4/8. Since we could not identify any potential ChemRs in the genomes of amphioxus (Branchiostoma floridae) and sea squirts (Ciona intestinalis), the genome of the jawless fish lamprey offers the best possibility of finding the primitive ChemR genes. In addition to the CXCR4 gene (Kuroda et al., 2003), there exist two pairs of ChemR 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, while Ccr_1 and Ccr_2 constitute a new group CCR14. CXCR4 and CXCR7 are known to play important roles in the mammalian and teleost development by binding CXCL12 as the signaling receptor and the scavenging receptor, respectively (Thelen and Thelen, 2008). Thus, the same set of ChemRs and CXCL12 may play a similar developmental role since the first appearance of the chemokine system in the lamprey. 7. Evolutionary history of the chemokine receptor genes Fig. 2 summarizes the gain and loss events of ChemR genes during the vertebrate evolution. Vertebrates are a group of chordate animals that contains the urochordates (sea 12

14 squirts) and cephalochordates (amphioxus). Since we and others (Bajoghli et al., 2009, DeVries et al., 2006) have failed to identify ChemRs in the genomes of amphioxus and sea squirts, agnathan lamprey is so far the most primitive vertebrate that possesses the ChemRs. In the lamprey, three CXCL genes (CXCL8-like, CXCL15-like and CXCL12) and two CCL chemokine genes have been identified (Bajoghli et al., 2009, Nomiyama et al., 2010). The two CCL chemokines may bind to the CCR14 group receptors, while CXCL12 is likely to bind to CXCR4 and the CXCR7 group (Cxcr_1 and Cxcr_2). Given that the receptor(s) for CXCL8-like and CXCL15-like chemokines are yet to be discovered, so the total of at least 5 ChemR groups may exist in the lamprey. The emergence of chemokine system in the lamprey coincides with the appearance of lymphocytes and adaptive immune responses in agnathans, although the immune system of agnathans is quite different from that of cartilaginous fishes (Cooper and Alder, 2006). Huising et al. have proposed that the original biological role of the CXC chemokines and their receptors might be in the central nervous system before they were adapted by the immune system (Huising et al., 2003a). However, this hypothesis may be disputable (Huising et al., 2003b, Shields, 2003). Given that the vertebrate nervous system originates from the basal chordate amphioxus (Holland, 2009) and the chemokines and their receptors are not yet found in amphioxus, the original role of the chemokine system might be rather related to the immune system in the ancient jawless vertebrates. It is noteworthy that the lamprey CXCR4 is expressed in lymphocyte-like cells (Kuroda et al., 2003). It is therefore of interest to know the types of cells expressing CCR14 and their biological role in lamprey. The entire genome is assumed to have been duplicated in two rounds at the dawn of vertebrate evolution (Meyer and Van de Peer, 2005, Sato and Nishida, 2010). One round of whole-genome duplication (1R) occurred before the divergence of jawless and jawed vertebrates, and the second one (2R) after the divergence of the jawless and jawed vertebrate lineages but before the split of cartilaginous fish and bony fish lineages (Venkatesh et al., 2007) (Fig. 2). An additional event (3R) leading to at least up to eight copies of the ancestral genome might have occurred in the stem lineage of ray-finned fishes after their divergence from the land vertebrates (Meyer and Van de Peer, 2005, Sato and Nishida, 2010). The presence of homeobox (Hox) gene clusters on four chromosomes in most vertebrates but only on one chromosome in invertebrates has been taken as the evidence for two-rounds of whole-genome duplication (Lundin et al., 2003). 13

15 Since two or more Hox-bearing chromosomes contain paralogs of a large number of gene families, these genes were assumed to be duplicated together with the Hox clusters by polyploidization. DeVries et al. (2006) proposed a model in which the ChemR genes increased their numbers by duplications concomitant with the Hox gene clusters. However, controversy still exists regarding the extent of the duplicated chromosome segments (Abbasi and Grzeschik, 2007, Hughes et al., 2001, Larhammar et al., 2002). The controversy may largely stem from too much dependence on the phylogenetic data due to the incomplete assemblies of the various animal genomes. Comparison of the genome maps of the regions spanning both the Hox clusters and ChemR genes, especially those of agnathans and cartilaginous fishes, may elucidate whether the ChemR genes were duplicated together with the Hox clusters by tetraploidization or translocated to some of the Hox-bearing chromosomes during evolution. The cartilaginous fish elephant shark and the tetrapods contain four Hox clusters, whereas the lamprey contains at least three Hox clusters, one of which seems to be the result of a lineage-specific duplication event (Force et al., 2002). Compared to the lamprey, which possesses 5 genes (3 ChemR groups), elephant shark contain 14 genes (12 ChemR groups). The presence of four times the number of ChemR groups in elephant shark as in lamprey suggests that ChemR duplication events might have occurred in the cartilaginous fish lineage besides the second whole-genome duplication. Furthermore, 9 of the 12 elephant shark ChemR groups are among the 11 homeostatic or dual function receptors in mammals. Among the rest of the three receptors (CXCR1 & 2, CCR12, and CXCR7), only CCR12 is not present in the mammals. These results suggest that the basic components of the ChemRs, in particular the homeostatic and dual function classes, have been well established before the divergence of cartilaginous and bony fishes. Compared to elephant shark, the teleost fishes contain more ChemR genes and groups. Although two ChemR groups (CXCR6 and CCR4) were lost in the teleosts, 6 novel groups were generated, making the total number of the ChemR groups to 16. Among the newly emerged ChemR groups are CXCR3, CXCR3L, and CCRL1, all of which have been transmitted to mammals and/or birds. The other 3 groups (CCR4 & 8, CCR11, and XCR1L) were teleost-specific and lost in the other lineages. There are total 24 and 31 ChemR genes in Tetraodon and Medaka, respectively. On the other hand, there are 41 genes in zebrafish. This may be in part due to the additional fish-specific whole-genome duplication, but local duplication events may also have contributed to the 14

16 increases. Since the appearance 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 in the mammalian lineage. There are also gene losses in the bird lineage. Similarly, some of the tetrapod-specific group genes might have been deleted in the lower vertebrates. For example, DARC is highly divergent from other ChemRs probably due to its ancient origin but only present in higher vertebrates. Eventually, mammals contain 23 groups (23 genes in human) compared to 12 groups (14 genes) in elephant shark. Since no whole-genome duplication event has occurred during the evolution from cartilaginous fishes to mammals, recurrent local gene duplications may have amplified the diversity of the ChemR system in mammals. As described above, most extant homeostatic and dual function ChemRs were generated at the early stages of vertebrate evolution. In contrast, most of the ChemR genes supposed to have inflammatory functions might not have been transmitted to higher vertebrates during the vertebrate evolution so that the mammalian inflammatory ChemRs have originated in amniotes. Such short life-spans of inflammatory ChemR genes may be in part due to the survival strategy used by host animals to avoid the viral mimicry of chemokines and their receptors (Murphy, 2001). One exception is the CXCR1 & 2 group genes, which have originated in jawless or cartilaginous fish and have been retained throughout the vertebrate evolution. From these considerations, it may not be necessarily true that the ancestor ChemR is a homeostatic one. It is therefore interesting to see which class the lamprey CCR14 group genes belong to and what class of ChemR is a prototype. 8. Conclusion The application of human nomenclature system to lower animals has caused a substantial confusion in the naming of vertebrate ChemR genes. We have therefore comprehensively surveyed the ChemR genes of 16 vertebrate species and classified the genes through the combination of phylogenetic and comparative genomic analyses. The results show that there are 29 ChemR orthologous groups in vertebrates and that the gain and loss of the groups occur as a continuous process in all lineages even after the whole-genome duplication events. Our orthologous grouping may provide the basis for a unified nomenclature of ChemR genes and may also be useful to understand the function of each ChemR group members. A rough evolutionary history of the ChemR genes is also 15

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23 Figure legends Fig. 1. Genomic organization of the human chemokine receptors. Arrows indicate the chemokine receptor genes and their transcriptional orientation. The chemokine ligands recognized by each receptor are also shown below the receptor genes. The signaling receptors for CXCL14, CXCL17, and CCL18 are as yet unknown. Fig. 2. Evolutionary history of the chemokine receptor orthologous groups. The divergence times (Mya, million years ago) shown are the minimum divergence times estimated based on fossil records (Benton and Donoghue, 2007, Janvier, 2006, Sansom et al., 1996). The timings of the two successive rounds of whole-genome duplication (1R and 2R) and the fish-specific genome duplication (3R) are also shown. The mammalian ChemRs present in the major gene cluster are indicated with asterisks. The elephant shark (cartilaginous fish) CCR4/8 gene is provisionally included in the CCR4 orthologous group. 22