1471-2164-9-222 1471-2164 Research article <p>Extensive expansion and diversification of the chemokine gene family in zebrafish: Identification of a novel chemokine subfamily CX</p> Nomiyama Hisayuki nomiyama@gpo.kumamoto-u.ac.jp Hieshima Kunio hieshima@med.kindai.ac.jp Osada Naoki nosada@nibio.go.jp Kato-Unoki Yoko ykato@agr.kyushu-u.ac.jp Otsuka-Ono Kaori kkjun3@yahoo.co.jp Takegawa Sumio zorro324@yahoo.co.jp Izawa Toshiaki f050301m@mbox.nagoya-u.ac.jp Yoshizawa Akio k070033@hiroshima-u.ac.jp Kikuchi Yutaka yutaka@hiroshima-u.ac.jp Tanase Sumio tanase@gpo.kumamoto-u.ac.jp Miura Retsu miura@gpo.kumamoto-u.ac.jp Kusuda Jun jkusuda@nibio.go.jp Nakao Miki miki_n@agr.kyushu-u.ac.jp Yoshie Osamu o.yoshie@med.kindai.ac.jp

Department of Molecular Enzymology, Kumamoto University Graduate School of Medical Sciences, Kumamoto 860-8556, Japan

Department of Microbiology, Kinki University School of Medicine, Osaka-Sayama, Osaka 589-8511, Japan

Department of Biomedical Resources, National Institute of Biomedical Innovation, Ibaraki, Osaka 567-0085, Japan

Laboratory of Marine Biochemistry, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, Fukuoka 812-8581, Japan

Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan

Department of Analytical Biochemistry, Kumamoto University School of Health Sciences, Kumamoto 862-0976, Japan

Department of Biological Science, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan

 BMC Genomics 1471-2164 2008 9 1 222 http://www.biomedcentral.com/1471-2164/9/222 18482437 10.1186/1471-2164-9-222 23 10 2007 15 5 2008 15 5 2008 2008 Nomiyama et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

The chemokine family plays important roles in cell migration and activation. In humans, at least 44 members are known. Based on the arrangement of the four conserved cysteine residues, chemokines are now classified into four subfamilies, CXC, CC, XC and CX3C. Given that zebrafish is an important experimental model and teleost fishes constitute an evolutionarily diverse group that forms half the vertebrate species, it would be useful to compare the zebrafish chemokine system with those of mammals. Prior to this study, however, only incomplete lists of the zebrafish chemokine genes were reported.

Results

We systematically searched chemokine genes in the zebrafish genome and EST databases, and identified more than 100 chemokine genes. These genes were CXC, CC and XC subfamily members, while no CX3C gene was identified. We also searched chemokine genes in pufferfish fugu and Tetraodon, and found only 18 chemokine genes in each species. The majority of the identified chemokine genes are unique to zebrafish or teleost fishes. However, several groups of chemokines are moderately similar to human chemokines, and some chemokines are orthologous to human homeostatic chemokines CXCL12 and CXCL14. Zebrafish also possesses a novel species-specific subfamily consisting of five members, which we term the CX subfamily. The CX chemokines lack one of the two N-terminus conserved cysteine residues but retain the third and the fourth ones. (Note that the XC subfamily only retains the second and fourth of the signature cysteines residues.) Phylogenetic analysis and genome organization of the chemokine genes showed that successive tandem duplication events generated the CX genes from the CC subfamily. Recombinant CXL-chr24a, one of the CX subfamily members on chromosome 24, showed marked chemotactic activity for carp leukocytes. The mRNA was expressed mainly during a certain period of the embryogenesis, suggesting its role in the zebrafish development.

Conclusion

The phylogenic and genomic organization analyses suggest that a substantial number of chemokine genes in zebrafish were generated by zebrafish-specific tandem duplication events. During such duplications, a novel chemokine subfamily termed CX was generated in zebrafish. Only two human chemokines CXCL12 and CXCL14 have the orthologous chemokines in zebrafish. The diversification observed in the numbers and sequences of chemokines in the fish may reflect the adaptation of the individual species to their respective biological environment.

Background

Chemokines are a family of chemotactic cytokines that regulate migration and tissue compartmentalization of various kinds of cells in the body 1234. Chemokine genes have been found only in vertebrates including jawless fish lamprey, the most primitive vertebrate 5. Based on the arrangement of the four conserved cysteine residues, chemokines are now classified into four subfamilies, CXC, CC, XC and CX3C. One or three amino acid residues separate the first and second cysteine residues in the CXC and CX3C chemokines, respectively. In the CC subfamily, the two N-terminus cysteines are juxtaposed. The XC subfamily (often called as C subfamily) lacks the first and third conserved cysteine residues. There are at least 44 chemokine genes (24 CC, 17 CXC, one CX3C, and two XC genes) in humans. In addition, there are four CC chemokine genes (CCL3L1, CCL3L3, CCL4L1, and CCL4L2), which are highly similar to CCL3 or CCL4 genes but their gene copy numbers are variable between individuals. In mice, there are 38 genes (21 CC, 15 CXCL, one CX3C, and one XC genes) in addition to several CC genes similar to Ccl19, Ccl21, and Ccl27 with variable copy numbers between strains. Besides gene number differences, there exist lineage-specific chemokine genes as well as genes ambiguous in the orthologous relationship even between human and mouse. On the other hand, only 18 functionally signaling chemokine receptors are identified in both human and mouse. Hence, multiple chemokines can bind to the same receptor.

Chemokine genes can be divided into two groups based on the genomic organization 3. The genes of one group are located in large clusters at particular chromosome locations (the major-cluster chemokines). In humans and mice, there are two major gene clusters consisting of CC genes and CXC genes, respectively. Another group of chemokines are located separately in unique chromosomal locations, singly or in mini-clusters. The major-cluster chemokines usually bind to multiple receptors and often do not correspond well between species. They were probably generated by multiple gene duplication events occurred relatively recently in the evolutionary terms. In contrast, mini-cluster or non-cluster chemokine genes are well conserved between the species and tend to act on a single receptor.

In addition to the protein or genome structure-based classifications, the chemokines can also be functionally classified into two major groups based on their mode of expression and function, the inflammatory and homeostatic chemokines 6. Inflammatory chemokines are expressed by leukocytes or related cells only upon activation and mediate emigration of leukocytes, while homeostatic chemokines are constitutively expressed in tissues such as lymphoid tissues and are involved in relocation of lymphocytes or other types of cells. Some chemokines are known to have both properties and are called dual-function chemokines. Notably, most homeostatic and dual-function chemokines belong to the mini- or non-cluster chemokines, while most inflammatory chemokines to the major cluster chemokines.

Teleost fishes constitute an evolutionarily diverse group and form half of the vertebrate species. The availability of genomic resources in the form of genome sequences and ESTs (Expressed Sequence Tags)/cDNAs for fishes that belong to different orders now allows an evolutionary analysis of the chemokine gene family in the fish. The draft genome sequences are available for several teleosts, and we chose zebrafish (Danio rerio) and two pufferfish species, Fugu rubripes (or Takifugu rubripes) 7 and Tetraodon nigroviridis 8, for this study. The genome size of zebrafish is 1,700 Mb, about half of the size of human genome, and those of the two pufferfish species are only 390 Mb. The divergence between the lineages leading to zebrafish and pufferfish was estimated to be about 150 million years (Myr) ago from the fossil data 910 or about 324 Myr ago from the molecular data 11. The two pufferfish species belong to the order Tetraodontiformes and diverged from a common ancestor approximately 32 Myr ago from the fossil data 10 or 85 Myr ago from the molecular data 11. The genome sequences of these fishes are nearly complete, and 85% (zebrafish) and 90% (the two pufferfish species) of the genomes are represented in the draft sequences.

Zebrafish is a useful experimental model to study various biological processes in vivo, particularly the developmental processes 121314. Previously, by using zebrafish, Doitsidou et al. 15 and Knaut et al. 16 have demonstrated that the CXC chemokine cxcl12a (sdf-1a) and its receptor cxcr4 are critical for proper migration of the primordial germ cells, the progenitors of the gametes. Furthermore, also by using zebrafish, the cxcl12-cxcr4 signaling has also been shown to be important for the migration of sensory cells known as the migrating primordium of the posterior lateral line as well as the guidance of retinal ganglion cell axons 1718. These finding have clearly demonstrated the usefulness of this model organism for studying the chemokine functions in the embryogenesis.

Recently, Peatman and Liu 19 reported identification of 46 CC chemokine genes in the zebrafish genome. They compared them with the 26 catfish CC chemokines that they previously identified, and showed evidence of extensive, species-specific intrachromosomal duplications. They also showed such gene duplications are widespread among the teleost fishes by analyzing CC chemokine genes of various species 19. DeVries et al. 20 also identified 127 vertebrate chemokine genes including 63 zebrafish genes and showed that the tandem and en bloc duplications have been used as means for selective expansion of the chemokine superfamily members. However, the zebrafish genome project is still ongoing, and the genome sequence that these two groups have used was the previous version. In the present study, we systematically searched for the chemokine genes in the latest zebrafish genome and EST databases. We have identified more than 100 chemokine genes in the zebrafish genome including a novel chemokine subfamily termed CX. Phylogenic and syntenic analyses with other fish and mammal chemokines have demonstrated that extensive and highly species-specific duplication events were involved in the evolution of the zebrafish chemokine system. We have further demonstrated that several chemokines including one of the CX subfamily show developmentally regulated expression patterns, suggesting their potential roles in the embryonic development of zebrafish.

Results

Chemokine gene prediction and isolation of chemokine cDNAs

Using various chemokine sequences as queries, we searched for chemokine genes in the zebrafish draft genome and EST databases and identified a total of 101 possible chemokine gene sequences (Table 1) in addition to the four chemokine genes reported previously by other groups, ccl1 (GenBank accession number NM_131062), cxcl12a and cxcl12b 15, and scyba 21. To determine whether the genes predicted from the genome sequence are expressed and also to confirm the exon-intron junctions, a mixture of cDNAs prepared from various developmental stages of zebrafish was used as a template for RT-PCR using primers located outside the coding sequences (Table 2). A total of 34 cDNAs were isolated. The 5' sequence of one clone (CCL-chrUi) was obtained by RACE reaction. Six of them were isolated incidentally due to their close similarity to the original cDNA sequences. Therefore, a total of 107 chemokine genes were newly identified, of which 71 genes have corresponding cDNAs or ESTs (Table 1). Considering the zebrafish draft sequence covers approximately 85% of the genome, these results suggest that there would possibly exist more than the currently identified 111 (4+107) chemokine genes in the zebrafish genome, far exceeding the number of the mammalian chemokine genes. Indeed, there are a number of chemokine-like exons located near or within the intact chemokine genes (not shown). Although most of them may be pseudogenes, the others might turn out to be intact genes if the gaps in the draft sequence are filled by the ongoing sequencing project.

<p>Table 1</p>

Chemokine genes in zebrafisha

Chemokin

Chr

Ori

Location

Exons b

GenBank

EST c

aa d

O-Glyco e

CCL-chr25a

25

+

19027505–19029332

3

AB331747

EH278089

85

0 (0)

CCL-chr25b

25

+

19035165–19036703

3

110

0 (0)

CCL-chr25c

25

+

19075615–19076539

3

99

0 (0)

CCL-chr25d

25

+

19078908–19089162

3

Dr.112407

96

0 (0)

CCL-chr25e

25

+

19090993–19094737

3

95

0 (0)

CCL-chr25f

25

+

19100444–19101671

3

Dr.89844

102

1 (2)

CCL-chr25g

25

+

19268675–19270505

3

Dr.111511

85

0 (0)

CCL-chr25h

25

+

19276303–19277760

3

109

0 (0)

CCL-chr25i

25

-

19352626–19353853

3

Dr.89844

102

1 (2)

CCL-chr25j

25

-

19359559–19363303

3

95

0 (0)

CCL-chr25k

25

-

19365134–19374852

3

96

0 (0)

CCL-chr25l

25

-

19376636–19377560

3

99

0 (0)

CCL-chr25m

25

-

19421398–19422248

3

201

60 (60)

CCL-chr25n

25

-

19435712–19436261

3

93

0 (0)

CCL-chr25o

25

-

19437496–19438019

3

AB331748

Dr.112677

94

0 (0)

CCL-chr25p

25

-

19457474–19458202

3

157

37 (37)

CCL-chr25q

25

-

19462780–19463308

3

94

1 (1)

CCL-chr25r

25

-

19466008–19467922

3

Dr.125717

87

0 (2)

CCL-chr25s

25

+

19503739–19505640

3

AB331749

Dr.125717

84

0 (1)

CCL-chr25t

25

+

19508322–19508850

3

AB331750

94

1 (1)

CCL-chr25u

25

+

19513230–19513960

3

158

37 (37)

CCL-chr25v

25

+

19536723–19537246

3

AB331751

94

0 (0)

CCL-chr25w

25

+

19538474–19539024

3

93

0 (0)

CCL-chr25x

25

+

19552761–19553847

3

277

104 (104)

CCL-chr25y

25

-

19582718–19584968

3

AB331752

Dr.93495

98

0 (0)

CCL-chr25z

25

-

20857424–20858639

3

Dr.131114

163

36 (36)

CCL-chr25aa

25

-

21213925–21215140

3

Dr.131114

163

36 (36)

CCL-chr25ab

25

-

21241306–21243245

3

AB331753

Dr.93495

98

1 (1)

CCL-chr25ac

25

-

21252391–21254330

3

AB331754

Dr.93495

98

1 (1)

CCL-chr24a

24

+

30502892–30505104

4

AB331755

DV590257

102

3 (3)

CXCL-chr24a

24

-

31852479–31853361

3

187

7 (7)

CXCL-chr24b

24

-

>31853500–31855624

3

153

9 (9)

CXCL-chr24c

24

-

31862084–31862883

3

Dr.92223

159

18 (18)

CXL-chr24a

24

+

32266050–32284596

5

AB331756

Dr.18825

115

0 (1)

CXL-chr24b

24

+

32290478–32291634

5

115

0 (0)

CCL-chr24b

24

+

32293590–32295005

5

AB331757

CO359348

116

0 (1)

CCL-chr24c

24

+

32302418–32304645

5

AB331758

Dr.130538

118

0 (0)

CCL-chr24d

24

+

32306481–32308480

5

CT653601

127

0 (0)

CCL-chr24e

24

+

32322636–32323565

5

118

0 (0)

CCL-chr24f

24

+

32329863–32331934

5

117

0 (0)

CXCL-chr24d

24

+

32345052–32346003

3

210

6 (6)

CXCL-chr24e

24

-

32352898–32353697

3

AB331759

Dr.92223

159

18 (18)

CCL-chr24g

24

-

32414552–32415181

3

Dr.111235

115

0 (0)

CCL-chr24h

24

-

32433297–32434055

4

120

2 (2)

CCL-chr24i

24

-

32440032–32440892

4

Dr.125570

135

17 (18)

CCL-chr24j

24

+

32451758–32453957

4

AB331760

Dr.897

98

0 (0)

CCL-chr24k

24

+

32461305–32462101

4

108

5 (5)

CCL-chr24l

24

+

32463704–32464522

4

106

7 (12)

CCL-chr24m

24

+

32470848–32473296

4

AB331761

Dr.29197

97

0 (2)

CCL-chr24n

24

+

33094971–33096928

5

117

0 (0)

CCL-chr23a

23

+

16185601–16187309

3

245

29 (29)

CXCL12bL

22

-

31836220–31849609

4

97

0 (0)

cxcl12b f

22

-

33091953–33105285

4

NM_198068

Dr.27045

97

0 (0)

CCL-chr20a

20

-

45279550–45280130

3

Dr.22573

103

0 (0)

CCL-chr20b

20

+

455913434–5591923

3

Dr.22573

103

0 (0)

CCL-chr20c

20

+

45594097-45594746

3

Dr.84529

103

0 (0)

CCL-chr20d

20

+

45600362–45600907

3

AB331762

EH279763

101

1 (1)

CCL-chr20e

20

+

45604369–45606327

5

EH547529

174

1 (1)

CCL-chr20f

20

+

45615627–45618919

5

163

3 (3)

CCL-chr20g

20

-

46647358–46648220

4

AB331763

106

0 (0)

CXL-chr19a

19

+

23008696–23011009

5

115

0 (1)

CCL-chr17a

17

-

43053286–43056335

4

AB331764

Dr.110765

111

7 (7)

CCL-chr17b

17

-

43061247–43065632

5

CT653600

127

0 (0)

CCL-chr17c

17

-

>43069244–43070260

(5)g

108

0 (1)

scyba f

14

-

27560158–27568603

4

NM_131627

Dr.117782

100

0 (0)

CXCL-chr13a

13

+

36660529–36661730

3

EH438452

90

0 (2)

CXCL-chr13b

13

+

36664145–36666791

4

Dr.88

120

0 (0)

cxcl12a f

13

-

36845066–36857673

4

NM_178307

Dr.99352

99

0 (0)

CXCL-chr13c

13

+

51125309–51128037

3

AB331765

Dr.82552

117

7 (8)

CXCL-chr13d

13

+

51138026–51141305

3

AB331766

Dr.92011

101

0 (0)

CXL-chr12a

12

+

23085158–23087450

5

Dr.32443

123

0 (2)

CCL-chr11a

11

+

5493062–5496539

3

AB331767

Dr.81010

106

0 (0)

CCL-chr11b

11

+

5570254–5575583

5

AB331768

Dr.85019

126

1 (2)

CCL-chr10a

10

-

13736505–13739228

4

Dr.92663

114

0 (1)

CCL-chr10b

10

+

(3289242)-48827709

3

AB331769

EE205697

91

0 (0)

ccl1 f

8

-

10307292–10323292

3

NM_131062

Dr.76247

93

0 (0)

CCL-chr7a

7

+

51890176–51890837

3

Dr.111629

93

0 (0)

CCL-chr5a

5

-

8165983–8172469

4

AB331770

Dr.86771

119

0 (0)

CCL-chr5b

5

-

8182370–8184709

3

EH281478

107

0 (0)

CXCL-chr5a

5

-

33964121–33965393

4

Dr.84656

115

2 (2)

CXCL-chr5b

5

-

33996816–33998000

4

Dr.111133

102

0 (0)

CXCL-chr5c

5

+

50933627–50940725

4

AB331771

CN175165

147

0 (0)

CXCL-chr5d

5

+

50944403–50945002

4

Dr.115041

94

0 (1)

CXCL-chr5e

5

+

50954047–50954534

4

Dr.89665

121

13 (13)

CXCL-chr5f

5

+

50959591–50960454

4

96

0 (2)

CXCL-chr5g

5

+

51761233–51761767

3

106

2 (2)

CXCL-chr5h

5

+

51766388–51766850

3

92

0 (0)

CXCL-chr5i

5

+

51772479–51772941

3

92

0 (0)

CCL-chr2a

2

-

38292712–38293705

3

247

47 (47)

CCL-chr2b

2

-

38310649–38315607

3

195

16 (16)

CCL-chr2c

2

-

38329197–38329990

3

Dr.113953

150

0 (0)

XCL-chr2a

2

-

38339304–38339976

3

EH453597

126

1 (3)

CCL-chr2d

2

+

38353445–38354111

4

Dr.86775

138

8 (8)

CCL-chr2e

2

+

38356664–38358727

4

AB331772

Dr.113952

111

4 (4)

CCL-chr2f

2

-

38369684–38371438

3

Dr.92436

89

0 (0)

CXCL-chr1a

1

+

14796681–14797310

4

98

0 (0)

CXCL-chr1b

1

+

15001965–15002594

4

98

0 (0)

CXCL-chr1c

1

-

61228824–61231793

4

AB331773

Dr.17289

114

0 (1)

CCL-chr1a

1

-

68219709–68220766

3

254

59 (59)

CCL-chr1b

1

-

68251400–68252095

3

130

0 (0)

CCL-chr1c

1

-

68253292–68254202

3

220

26 (26)

CCL-chrUa

U

AB331774

EH599800

99

0 (0)

CCL-chrUb

U

AB331775

108

0 (0)

CCL-chrUc

U

AB331776

99

0 (0)

CCL-chrUd

U

AB331777

94

1 (1)

CCL-chrUe

U

AB331778

110

0 (0)

CCL-chrUf

U

Dr.83347

144

6 (6)

CCL-chrUg

U

CK237158

131

3 (3)

CCL-chrUh

U

EH479986

95

0 (0)

CCL-chrUi

U