NORMAL GENOMIC

• Uusi-Heikkila S et al.
• The evolutionary legacy of size-selective harvesting extends from genes to populations.
• Evol Appl. 2015; 8: 597-620
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• Size-selective harvesting is assumed to alter life histories of exploited fish populations, thereby negatively affecting population productivity, recovery, and yield. However, demonstrating that fisheries-induced phenotypic changes in the wild are at least partly genetically determined has proved notoriously difficult. Moreover, the population-level consequences of fisheries-induced evolution are still being controversially discussed. Using an experimental approach, we found that five generations of size-selective harvesting altered the life histories and behavior, but not the metabolic rate, of wild-origin zebrafish (Danio rerio). Fish adapted to high positively size selective fishing pressure invested more in reproduction, reached a smaller adult body size, and were less explorative and bold. Phenotypic changes seemed subtle but were accompanied by genetic changes in functional loci. Thus, our results provided unambiguous evidence for rapid, harvest-induced phenotypic and evolutionary change when harvesting is intensive and size selective. According to a life-history model, the observed life-history changes elevated population growth rate in harvested conditions, but slowed population recovery under a simulated moratorium. Hence, the evolutionary legacy of size-selective harvesting includes populations that are productive under exploited conditions, but selectively disadvantaged to cope with natural selection pressures that often favor large body size.

• Gratacap RL, Wheeler RT
• Utilization of zebrafish for intravital study of eukaryotic pathogen-host interactions.
• Dev Comp Immunol. 2014; 46: 108-15
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• Unique imaging tools and practical advantages have made zebrafish a popular model to investigate in vivo host-pathogen interactions. These studies have uncovered details of the mechanisms involved in several human infections. Until recently, studies using this versatile host were limited to viral and prokaryotic pathogens. Eukaryotic pathogens are a diverse group with a major impact on the human and fish populations. The relationships of eukaryote pathogens with their hosts are complex and many aspects remain obscure. The small and transparent zebrafish, with its conserved immune system and amenability to genetic manipulation, make it an exciting model for quantitative study of the core strategies of eukaryotic pathogens and their hosts. The only thing to do now is realize its potential for advancement of biomedical and aquaculture research.

• Hu YW, Yu ZL, Xue NN, Nie P, Chang MX
• Expression and protective role of two novel NACHT-containing proteins in pathogen infection.
• Dev Comp Immunol. 2014; 46: 323-32
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• Lower vertebrates have been found to possess over 200 NACHT-domain encoding genes; but, to date, very little is known about their functional activity. This article describes the sequences and expression analysis of two zebrafish NACHT-containing proteins, namely NALPL1 and NALPL2. In addition, the functions of zebrafish NALPL1 and NALPL2, which are absent for both amino-terminal effector-binding domain (EBD) and carboxy-terminal ligand-recognition domain (LRD), were investigated for the first time in fish species. The predicted NALPL1 and NALPL2 proteins consist of 651 and 847 amino acids (aa), respectively, with both molecules only containing NACHT domain, which were different from other NACHT-family members. Phylogenetic analysis showed that zebrafish NALPL1 and NALPL2 have a closer relationship with mammalian NALP subfamily than NOD subfamily. The differential expression patterns of NALPL1 and NALPL2 in development stages and organs were observed, suggesting the difference of action phase and effector organ of NALPL1 and NALPL2. When the modulation of NALPL1 and NALPL2 in pathogen infection was analyzed, it was found that the two molecules were upregulated by both bacterial and viral infection. Overexpression of NALPL1 and NALPL2 resulted in significant inhibition for intracellular Edwardsiella tarda growth. Further studies demonstrated that NALPL1 and NALPL2 also contributed to protection against viral infection. These results demonstrate that both NALPL1 and NALPL2 are important intracellular proteins in host surveillance against both bacterial and viral infection. Interestingly, the expression of downstream signaling genes was not affected by the overexpression of NALPL1 or NALPL2, but NOD1 and MDA5 were upregulated by NALPL1 or NALPL2 overexpression, suggesting that they likely act in pathogen infection through the interaction with other PRRs.

• Hamada M, Shoguchi E, Shinzato C, Kawashima T, Miller DJ, Satoh N
• The complex NOD-like receptor repertoire of the coral Acropora digitifera includes novel domain combinations.
• Mol Biol Evol. 2013; 30: 167-76
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• Innate immunity in corals is of special interest not only in the context of self-defense but also in relation to the establishment and collapse of their obligate symbiosis with dinoflagellates of the genus Symbiodinium. In innate immunity system of vertebrates, approximately 20 tripartite nucleotide oligomerization domain (NOD)-like receptor proteins that are defined by the presence of a NAIP, CIIA, HET-E and TP1 (NACHT) domain, a C-terminal leucine-rich repeat (LRR) domain, and one of three types of N-terminal effector domain, are known to function as the primary intracellular pattern recognition molecules. Surveying the coral genome revealed not only a larger number of NACHT- and related domain nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 (NB-ARC)-encoding loci (~500) than in other metazoans but also surprising diversity of domain combinations among the coral NACHT/NB-ARC-containing proteins; N-terminal effector domains included the apoptosis-related domains caspase recruitment domain (CARD), death effector domain (DED), and Death, and C-terminal repeat domains included LRRs, tetratricopeptide repeats, ankyrin repeats, and WD40 repeats. Many of the predicted coral proteins that contain a NACHT/NB-ARC domain also contain a glycosyl transferase group 1 domain, a novel domain combination first found in metazoans. Phylogenetic analyses suggest that the NACHT/NB-ARC domain inventories of various metazoan lineages, including corals, are largely products of lineage-specific expansions. Many of the NACHT/NB-ARC loci are organized in pairs or triplets in the Acropora genome, suggesting that the large coral NACHT/NB-ARC repertoire has been generated at least in part by tandem duplication. In addition, shuffling of N-terminal effector domains may have occurred after expansions of specific NACHT/NB-ARC-repeat domain types. These results illustrate the extraordinary complexity of the innate immune repertoire of corals, which may in part reflect adaptive evolution to a symbiotic lifestyle in a uniquely complex and challenging environment.

• Howe K et al.
• The zebrafish reference genome sequence and its relationship to the human genome.
• Nature. 2013; 496: 498-503
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• Zebrafish have become a popular organism for the study of vertebrate gene function. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.

• Sundaram AY, Consuegra S, Kiron V, Fernandes JM
• Positive selection pressure within teleost Toll-like receptors tlr21 and tlr22 subfamilies and their response to temperature stress and microbial components in zebrafish.
• Mol Biol Rep. 2012; 39: 8965-75
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• Toll-like receptors (TLRs) play a crucial role in host defence, since they trigger immune response following recognition of pathogen-associated molecular patterns (PAMPs) in potential infectious agents. TLRs have been found in numerous organisms, including mammals, birds and teleosts. Some TLR members are commonly retained across all species, whilst others were lost, gained or diverged independently during evolution. Our knowledge about the evolution and specific functions of tlr21, tlr22 and tlr23 in teleosts are still scarce. Phylogenetic analysis of 18 tlr13, tlr21, tlr22 and tlr23 genes from 9 different fish species divided them in two groups. All tlr21 genes were under the first clade, while the second comprised tlr22, tlr23 and tlr13 from Atlantic salmon. Evidence of positive selection was detected at three sites within the leucine-rich repeat regions of Tlr22, which may influence PAMP recognition. Immunostimulation experiments revealed that expression of zebrafish tlr22 is modulated by several unrelated PAMPs. Up to a 3-fold increase in tlr21 and tlr22 expression was detected in larvae exposed to immunostimulants such as lipopolysaccharide, peptidoglycan or poly I:C. We found that zebrafish tlrs are expressed mainly in immune-related organs, such as spleen and kidney as well as in testis and temperature stress did not have an effect on the expression of tlr21 and tlr22 in the early stages of development in zebrafish larvae. Our data indicates that these teleost tlrs may play a role in innate host defence. In particular, tlr22 is evolving under positive selection, which indicates functional diversification and adaptation of the response to different PAMPs.

• Ordas A et al.
• Deep sequencing of the innate immune transcriptomic response of zebrafish embryos to Salmonella infection.
• Fish Shellfish Immunol. 2011; 31: 716-24
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• Salmonella enterica serovar Typhimurium (S. typhimurium) bacteria cause an inflammatory and lethal infection in zebrafish embryos. To characterize the embryonic innate host response at the transcriptome level, we have extended and validated previous microarray data by Illumina next-generation sequencing analysis. We obtained 10 million sequence reads from control and Salmonella-infected zebrafish embryos using a tag-based sequencing method (DGE or Tag-Seq) and 15 million reads using whole transcript sequencing (RNA-Seq), which respectively mapped to circa 65% and 85% of 28,716 known Ensembl transcripts. Both sequencing methods showed a strong correlation of sequence read counts per transcript and an overlap of 241 transcripts differentially expressed in response to infection. A lower overlap of 165 transcripts was observed with previous microarray data. Based on the combined sequencing-based and microarray-based transcriptome data we compiled an annotated reference set of infection-responsive genes in zebrafish embryos, encoding transcription factors, signal transduction proteins, cytokines and chemokines, complement factors, proteins involved in apoptosis and proteolysis, proteins with anti-microbial activities, as well as many known or novel proteins not previously linked to the immune response. Furthermore, by comparison of the deep sequencing data of S. typhimurium infection in zebrafish embryos with previous deep sequencing data of Mycobacterium marinum infection in adult zebrafish we derived a common set of infection-responsive genes. This gene set consists of known and putative innate host defense genes that are expressed both in the absence and presence of a fully developed adaptive immune system and that provide a valuable reference for future studies of host-pathogen interactions using zebrafish infection models.

• van Soest JJ, Stockhammer OW, Ordas A, Bloemberg GV, Spaink HP, Meijer AH
• Comparison of static immersion and intravenous injection systems for exposure of zebrafish embryos to the natural pathogen Edwardsiella tarda.
• BMC Immunol. 2011; 12: 58-58
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• BACKGROUND: The zebrafish embryo is an important in vivo model to study the host innate immune response towards microbial infection. In most zebrafish infectious disease models, infection is achieved by micro-injection of bacteria into the embryo. Alternatively, Edwardsiella tarda, a natural fish pathogen, has been used to treat embryos by static immersion. In this study we used transcriptome profiling and quantitative RT-PCR to analyze the immune response induced by E. tarda immersion and injection. RESULTS: Mortality rates after static immersion of embryos in E. tarda suspension varied between 25-75%, while intravenous injection of bacteria resulted in 100% mortality. Quantitative RT-PCR analysis on the level of single embryos showed that expression of the proinflammatory marker genes il1b and mmp9 was induced only in some embryos that were exposed to E. tarda in the immersion system, whereas intravenous injection of E. tarda led to il1b and mmp9 induction in all embryos. In addition, microarray expression profiles of embryos subjected to immersion or injection showed little overlap. E. tarda-injected embryos displayed strong induction of inflammatory and defense genes and of regulatory genes of the immune response. E. tarda-immersed embryos showed transient induction of the cytochrome P450 gene cyp1a. This gene was also induced after immersion in Escherichia coli and Pseudomonas aeruginosa suspensions, but, in contrast, was not induced upon intravenous E. tarda injection. One of the rare common responses in the immersion and injection systems was induction of irg1l, a homolog of a murine immunoresponsive gene of unknown function. CONCLUSIONS: Based on the differences in mortality rates between experiments and gene expression profiles of individual embryos we conclude that zebrafish embryos cannot be reproducibly infected by exposure to E. tarda in the immersion system. Induction of il1b and mmp9 was consistently observed in embryos that had been systemically infected by intravenous injection, while the early transcriptional induction of cyp1a and irg1l in the immersion system may reflect an epithelial or other tissue response towards cell membrane or other molecules that are shed or released by bacteria. Our microarray expression data provide a useful reference for future analysis of signal transduction pathways underlying the systemic innate immune response versus those underlying responses to external bacteria and secreted virulence factors and toxins.

• Boudinot P et al.
• Origin and evolution of TRIM proteins: new insights from the complete TRIM repertoire of zebrafish and pufferfish.
• PLoS One. 2011; 6: 22022-22022
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• Tripartite motif proteins (TRIM) constitute a large family of proteins containing a RING-Bbox-Coiled Coil motif followed by different C-terminal domains. Involved in ubiquitination, TRIM proteins participate in many cellular processes including antiviral immunity. The TRIM family is ancient and has been greatly diversified in vertebrates and especially in fish. We analyzed the complete sets of trim genes of the large zebrafish genome and of the compact pufferfish genome. Both contain three large multigene subsets--adding the hsl5/trim35-like genes (hltr) to the ftr and the btr that we previously described--all containing a B30.2 domain that evolved under positive selection. These subsets are conserved among teleosts. By contrast, most human trim genes of the other classes have only one or two orthologues in fish. Loss or gain of C-terminal exons generated proteins with different domain organizations; either by the deletion of the ancestral domain or, remarkably, by the acquisition of a new C-terminal domain. Our survey of fish trim genes in fish identifies subsets with different evolutionary dynamics. trims encoding RBCC-B30.2 proteins show the same evolutionary trends in fish and tetrapods: they evolve fast, often under positive selection, and they duplicate to create multigenic families. We could identify new combinations of domains, which epitomize how new trim classes appear by domain insertion or exon shuffling. Notably, we found that a cyclophilin-A domain replaces the B30.2 domain of a zebrafish fintrim gene, as reported in the macaque and owl monkey antiretroviral TRIM5alpha. Finally, trim genes encoding RBCC-B30.2 proteins are preferentially located in the vicinity of MHC or MHC gene paralogues, which suggests that such trim genes may have been part of the ancestral MHC.

• Hu YL, Zhu LY, Xiang LX, Shao JZ
• Discovery of an unusual alternative splicing pathway of the immunoglobulin heavy chain in a teleost fish, Danio rerio.
• Dev Comp Immunol. 2011; 35: 253-7
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• In present study, we identified a novel membrane immunoglobulin M isotype from zebrafish (Danio rerio), which was designated as mIgM-2, adding a new member to the Immunoglobulin family in teleost fish. The full length of cloned mIgM-2 cDNA was 611 bp, encoding 150 amino acids. The putative mIgM-2 protein sequence consists of one constant region and a trans-membrane region. Phylogenetic analysis showed that mIgM-2 grouped with the known zebrafish IgM sequences. The mIgM-2 mRNA was widely expressed in immune-related tissues including intestine, kidney and skin. In vivo stimulation with LPS significantly up-regulates the expression of mIgM-2. Our results will add new insight into the immunoglobulin class diversity of teleost fish, and to better understand the evolutionary history of adaptive immunity from fish to mammals as a whole.

• Aggad D et al.
• In vivo analysis of Ifn-gamma1 and Ifn-gamma2 signaling in zebrafish.
• J Immunol. 2010; 185: 6774-82
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• The zebrafish genome contains a large number of genes encoding potential cytokine receptor genes as judged by homology to mammalian receptors. The sequences are too divergent to allow unambiguous assignments of all receptors to specific cytokines, and only a few have been assigned functions by functional studies. Among receptors for class II helical cytokines-i.e., IFNs that include virus-induced Ifns (Ifn-) and type II Ifns (Ifn-gamma), together with Il-10 and its related cytokines (Il-20, Il-22, and Il-26)-only the Ifn--specific complexes have been functionally identified, whereas the receptors for the two Ifn-gamma (Ifn-gamma1 and Ifn-gamma2) are unknown. In this work, we identify conditions in which Ifn-gamma1 and Ifn-gamma2 (also called IFNG or IFN-gamma and IFN-gammarel) are induced in fish larvae and adults. We use morpholino-mediated loss-of-function analysis to screen candidate receptors and identify the components of their receptor complexes. We find that Ifn-gamma1 and Ifn-gamma2 bind to different receptor complexes. The receptor complex for Ifn-gamma2 includes cytokine receptor family B (Crfb)6 together with Crfb13 and Crfb17, whereas the receptor complex for Ifn-gamma1 does not include Crfb6 or Crfb13 but includes Crfb17. We also show that of the two Jak2 paralogues present in the zebrafish Jak2a but not Jak2b is involved in the intracellular transmission of the Ifn-gamma signal. These results shed new light on the evolution of the Ifn-gamma signaling in fish and tetrapods and contribute toward an integrated view of the innate immune regulation in vertebrates.

• Pruvot B, Laurens V, Salvadori F, Solary E, Pichon L, Chluba J
• Comparative analysis of nonaspanin protein sequences and expression studies in zebrafish.
• Immunogenetics. 2010; 62: 681-99
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• Nonaspanins constitute a family of proteins, also called TM9SF, characterized by a large non-cytoplasmic domain and nine putative transmembrane domains. This family is highly conserved through evolution and comprises three members in Saccharomyces cerevisiae, Dictyostelium discoideum, and Drosophila melanogaster, and four members are reported in mammals (TM9SF1-TM9SF4). Genetic studies in Dictyostelium and Drosophila have shown that TM9SF members are required for adhesion and phagocytosis in innate immune response, furthermore, human TM9SF1 plays a role in the regulation of autophagy and human TM9SF4 in tumor cannibalism. Here we report that the zebrafish genome encodes five members of this family, TM9SF1-TM9SF5, which show high level of sequence conservation with the previously reported members. Expression analysis in zebrafish showed that all members are maternally expressed and continue to be present throughout embryogenesis to adults. Gene expression could not be regulated by pathogen-associated molecular patterns such as LPS, CpG, or Poly I:C. By bioinformatic analyses of 80 TM9SF protein sequences from yeast, plants, and animals, we confirmed a very conserved protein structure. An evolutionary conserved immunoreceptor tyrosine-based inhibition motif has been detected in the cytoplasmic domain between transmembrane domain (TM) 7 and TM8 in TM9SF1, TM9SF2, TM9SF4 and TM9SF5, and at the extreme C-terminal end of TM9SF4. Finally, a conserved TRAF2 binding domain could also be predicted in the cytoplasmic regions of TM9SF2, TM9SF3, TM9SF4, and TM9SF5. This confirms the hypothesis that TM9SF proteins may play a regulatory role in a specific and ancient cellular mechanism that is involved in innate immunity.

• Gong YF, Xiang LX, Shao JZ
• CD154-CD40 interactions are essential for thymus-dependent antibody production in zebrafish: insights into the origin of costimulatory pathway in helper T cell-regulated adaptive immunity in early vertebrates.
• J Immunol. 2009; 182: 7749-62
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• The CD154-CD40-mediated costimulatory pathway is critical for T-B cell cooperation in thymus-dependent (TD) immune response in mammals. However, little is known about its existence and occurrence in lower vertebrates. Here, we report on the identification and functional characterization of CD154 and CD40 homologs from the zebrafish (Danio rerio) model. Zebrafish CD154 is a type II membrane-bound protein with a TNF homology domain in its extracellular C-terminal region, whose tertiary structure is a sandwich containing two stacked sheets with "jelly roll" topology, just as the human TNF members do. The zebrafish CD40 is a type I membrane-bound protein with a sequence pattern of four cysteine-rich domains in its extracellular N-terminal region. The consensus TNFR-associated factor (TRAF)2- and TRAF6-binding motifs in mammalian CD40 are found in the cytoplasmic tail of zebrafish CD40, which indicates similar signal transduction mechanisms to higher vertebrates. Zebrafish CD154 and CD40 are widely distributed and can be up-regulated by thymus-dependent Ag. The production of IgM was dramatically decreased by anti-CD154 or soluble CD40, and it was enhanced by soluble CD154 or CD154-encoding plasmid in vivo. Thymus-dependent Ag-induced CD154 expression was inhibited by cyclosporin A, suggesting that CD154 functionally associates with T cells. Double immunofluorescence staining showed that CD40 and membrane IgM colocalized in B cells. CD154-CD40 binding assays showed that CD154 specifically binds to CD40 at homodimeric form. Our results provide the first evidence for the existence of the functional CD154-CD40-mediated costimulatory pathway and helper T cell regulatory mechanism underlying adaptive immunity in a fish species.

• Mei J, Gui J
• Bioinformatic identification of genes encoding C1q-domain-containing proteins in zebrafish.
• J Genet Genomics. 2008; 35: 17-24
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• C1q is the first subcomponent of classical pathway in the complement system and a major link between innate and acquired immunities. The globular (gC1q) domain similar with C1q was also found in many non-complement C1q-domain-containing (C1qDC) proteins which have similar crystal structure to that of the multifunctional tumor necrosis factor (TNF) ligand family, and also have diverse functions. In this study, we identified a total of 52 independent gene sequences encoding C1q-domain-containing proteins through comprehensive searches of zebrafish genome, cDNA and EST databases. In comparison to 31 orthologous genes in human and different numbers in other species, a significant selective pressure was suggested during vertebrate evolution. Domain organization of C1q-domain-containing (C1qDC) proteins mainly includes a leading signal peptide, a collagen-like region of variable length, and a C-terminal C1q domain. There are 11 highly conserved residues within the C1q domain, among which 2 are invariant within the zebrafish gene set. A more extensive database searches also revealed homologous C1qDC proteins in other vertebrates, invertebrates and even bacterium, but no homologous sequences for encoding C1qDC proteins were found in many species that have a more recent evolutionary history with zebrafish. Therefore, further studies on C1q-domain-containing genes among different species will help us understand evolutionary mechanism of innate and acquired immunities.

• Laing KJ, Purcell MK, Winton JR, Hansen JD
• A genomic view of the NOD-like receptor family in teleost fish: identification of a novel NLR subfamily in zebrafish.
• BMC Evol Biol. 2008; 8: 42-42
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• BACKGROUND: A large multigene family of NOD-like receptor (NLR) molecules have been described in mammals and implicated in immunity and apoptosis. Little information, however, exists concerning this gene family in non-mammalian taxa. This current study, therefore, provides an in-depth investigation of this gene family in lower vertebrates including extensive phylogenetic comparison of zebrafish NLRs with orthologs in tetrapods, and analysis of their tissue-specific expression. RESULTS: Three distinct NLR subfamilies were identified by mining genome databases of various non-mammalian vertebrates; the first subfamily (NLR-A) resembles mammalian NODs, the second (NLR-B) resembles mammalian NALPs, while the third (NLR-C) appears to be unique to teleost fish. In zebrafish, NLR-A and NLR-B subfamilies contain five and six genes respectively. The third subfamily is large, containing several hundred NLR-C genes, many of which are predicted to encode a C-terminal B30.2 domain. This subfamily most likely evolved from a NOD3-like molecule. Gene predictions for zebrafish NLRs were verified using sequence derived from ESTs or direct sequencing of cDNA. Reverse-transcriptase (RT)-PCR analysis confirmed expression of representative genes from each subfamily in selected tissues. CONCLUSION: Our findings confirm the presence of multiple NLR gene orthologs, which form a large multigene family in teleostei. Although the functional significance of the three major NLR subfamilies is unclear, we speculate that conservation and abundance of NLR molecules in all teleostei genomes, reflects an essential role in cellular control, apoptosis or immunity throughout bony fish.

• Nomiyama H et al.
• Extensive expansion and diversification of the chemokine gene family in zebrafish: identification of a novel chemokine subfamily CX.
• BMC Genomics. 2008; 9: 222-222
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• 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.

• Ohtani M, Hayashi N, Hashimoto K, Nakanishi T, Dijkstra JM
• Comprehensive clarification of two paralogous interleukin 4/13 loci in teleost fish.
• Immunogenetics. 2008; 60: 383-97
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• Interleukins 4 and 13 (IL-4 and IL-13) are related cytokines important for Th2 immune responses and encoded by adjacent genes on human chromosome 5. Efforts were made previously to detect these genes in fish, but research was hampered by a lack of sequence conservation. A Tetraodon nigrovirides (green spotted pufferfish) gene was annotated as IL-4 by Li et al. (Mol Immunol, 44:2078-2086, 2007), but this annotation was not well substantiated. However, the present study concludes that the reported pufferfish gene belongs to the IL-4/13 lineage indeed, while also describing an additional IL-4/13 copy in a paralogous genomic region. Our analyses of IL-4/13 loci in fish describe (1) genomic region history, (2) characteristic intron-exon organization, (3) deduced IL-4/13 molecules for several teleost fish species, (4) IL-4/13 lineage-specific protein motifs including a cysteine pair (pair 1), and (5) computer software predictions of a type I cytokine fold. Teleost IL-4/13 molecules have an additional cysteine pair (pair 2) or remnants thereof, which is absent in mammalian IL-4 and IL-13. We were unable to determine if the teleost IL-4/13 genes are orthologous to either IL-4 or IL-13, or if these mammalian genes separated later in evolution.

• Nayak AS, Lage CR, Kim CH
• Effects of low concentrations of arsenic on the innate immune system of the zebrafish (Danio rerio).
• Toxicol Sci. 2007; 98: 118-24
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• Arsenic has been associated with a multitude of human health problems; however, its impact on host resistance to infection has not been extensively researched. In vertebrates, the innate immune response is vital for potentiating the adaptive immune response. Therefore, dampening of the innate immune response results in an immunocompromised host. In this present study, effects of low concentrations of arsenic on zebrafish resistance to infection are evaluated. Exposure to 2 and 10 ppb arsenic, both considered safe levels in drinking water, resulted in a greater than 50-fold increase in viral load and at least a 17-fold increase in bacterial load in embryos. To determine the cause of this amplified pathogen load, important components of the innate immune system were analyzed. Presence of arsenic dampened the overall innate immune health of the fish as evidenced by reductions in respiratory burst activity. Viral infection, after arsenic exposure, showed decreases of up to 13- and 1.5-fold changes in interferon and Mx mRNA expression, respectively. Bacterial infection, post arsenic exposure, demonstrated at least 2.5- and 4-fold declines in interleukin-1beta and tumor necrosis factor-alpha mRNA levels, respectively. Maximum expression of these essential cytokines was also delayed upon arsenic exposure. Our data indicate that arsenic exposure, at concentrations deemed safe in drinking water, suppresses the overall innate immune function in zebrafish and present the zebrafish as a unique model for studying immunotoxicity of environmental toxicants. To our knowledge, this is the first report describing the effects of such low levels of arsenic on host resistance to infection.

• Lin B et al.
• Acute phase response in zebrafish upon Aeromonas salmonicida and Staphylococcus aureus infection: striking similarities and obvious differences with mammals.
• Mol Immunol. 2007; 44: 295-301
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• Zebrafish has emerged as a valuable model for immunological studies. However, little is known about the overall picture of its immune response to infectious pathogens. Here we present the first systematic study of its immune response to Aeromonas salmonicida and Staphylococcus aureus, a Gram-negative and a Gram-positive bacteria, respectively. Genes induced upon infection were identified with suppression subtractive hybridization, with many of them encoding acute phase proteins (APPs). When compared with mammals, striking similarities and obvious differences have been observed. Both similar APPs (SAA, hepcidin and haptoglobin, etc.) and a similar system for the induction of APPs (which involves the TLRs, pro-inflammatory cytokines and C/EBPs) were identified, implying evolutionary conserved mechanisms among fish and mammals. Some novel APPs were also discovered, suggesting different immune strategies adopted by fish species. Among which, LECT2 was induced by up to 1000-fold upon infection, shedding new lights on the function of this gene. Our results constitute the first demonstration of a similar while different immune response in zebrafish and open new avenues for the investigation of evolutionary conserved and fish specific mechanisms of innate immunity.

• Liongue C, Ward AC
• Evolution of Class I cytokine receptors.
• BMC Evol Biol. 2007; 7: 120-120
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• BACKGROUND: The Class I cytokine receptors have a wide range of actions, including a major role in the development and function of immune and blood cells. However, the evolution of the genes encoding them remains poorly understood. To address this we have used bioinformatics to analyze the Class I receptor repertoire in sea squirt (Ciona intestinalis) and zebrafish (Danio rerio). RESULTS: Only two Class I receptors were identified in sea squirt, one with homology to the archetypal GP130 receptor, and the other with high conservation with the divergent orphan receptor CLF-3. In contrast, 36 Class I cytokine receptors were present in zebrafish, including representative members for each of the five structural groups found in mammals. This allowed the identification of 27 core receptors belonging to the last common ancestor of teleosts and mammals. CONCLUSION: This study suggests that the majority of diversification of this receptor family occurred after the divergence of urochordates and vertebrates approximately 794 million years ago (MYA), but before the divergence of ray-finned from lobe-finned fishes around 476 MYA. Since then, only relatively limited lineage-specific diversification within the different Class I receptor structural groups has occurred.

• Sullivan C, Postlethwait JH, Lage CR, Millard PJ, Kim CH
• Evidence for evolving Toll-IL-1 receptor-containing adaptor molecule function in vertebrates.
• J Immunol. 2007; 178: 4517-27
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• In mammals, Toll-IL-1R-containing adaptor molecule 1 (TICAM1)-dependent TLR pathways induce NF-kappaB and IFN-beta responses. TICAM1 activates NF-kappaB through two different pathways involving its interactions with TNFR-associated factor 6 and receptor-interacting protein 1. It also activates IFN regulatory factor 3/7 through its interaction with TANK-binding kinase-1, leading to the robust up-regulation of IFN-beta. In this study, we describe the role of zebrafish (Danio rerio) TICAM1 in activating NF-kappaB and zebrafish type I IFN. Zebrafish IFN is unique in that it cannot be categorized as being alpha- or beta-like. Through comprehensive sequence, phylogenetic, and syntenic analyses, we fully describe the identification of a zebrafish TICAM1 ortholog. Zebrafish TICAM1 exhibits sequence divergence from its mammalian orthologs and our data demonstrate that these sequence differences have functional consequences. Zebrafish TICAM1 activates zebrafish IFN; however, it does so in an apparently IFN regulatory factor 3/7-independent manner. Furthermore, zebrafish TICAM1 does not interact with zebrafish TNFR-associated factor 6, thus NF-kappaB activation is dependent upon its interaction with receptor-interacting protein 1. Comparative genome analysis suggests that TICAM1 and TICAM2 evolved from a common vertebrate TICAM ancestor following a gene duplication event and that TICAM2 was lost in teleosts following the divergence of the rayfin and lobefin fishes 450 million years ago. These studies provide evidence, for the first time, of the evolving function of a vertebrate TLR pathway.

• Hibino T et al.
• The immune gene repertoire encoded in the purple sea urchin genome.
• Dev Biol. 2006; 300: 349-65
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• Echinoderms occupy a critical and largely unexplored phylogenetic vantage point from which to infer both the early evolution of bilaterian immunity and the underpinnings of the vertebrate adaptive immune system. Here we present an initial survey of the purple sea urchin genome for genes associated with immunity. An elaborate repertoire of potential immune receptors, regulators and effectors is present, including unprecedented expansions of innate pathogen recognition genes. These include a diverse array of 222 Toll-like receptor (TLR) genes and a coordinate expansion of directly associated signaling adaptors. Notably, a subset of sea urchin TLR genes encodes receptors with structural characteristics previously identified only in protostomes. A similarly expanded set of 203 NOD/NALP-like cytoplasmic recognition proteins is present. These genes have previously been identified only in vertebrates where they are represented in much lower numbers. Genes that mediate the alternative and lectin complement pathways are described, while gene homologues of the terminal pathway are not present. We have also identified several homologues of genes that function in jawed vertebrate adaptive immunity. The most striking of these is a gene cluster with similarity to the jawed vertebrate Recombination Activating Genes 1 and 2 (RAG1/2). Sea urchins are long-lived, complex organisms and these findings reveal an innate immune system of unprecedented complexity. Whether the presumably intense selective processes that molded these gene families also gave rise to novel immune mechanisms akin to adaptive systems remains to be seen. The genome sequence provides immediate opportunities to apply the advantages of the sea urchin model toward problems in developmental and evolutionary immunobiology.

• Nonaka M, Kimura A
• Genomic view of the evolution of the complement system.
• Immunogenetics. 2006; 58: 701-13
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• The recent accumulation of genomic information of many representative animals has made it possible to trace the evolution of the complement system based on the presence or absence of each complement gene in the analyzed genomes. Genome information from a few mammals, chicken, clawed frog, a few bony fish, sea squirt, fruit fly, nematoda and sea anemone indicate that bony fish and higher vertebrates share practically the same set of complement genes. This suggests that most of the gene duplications that played an essential role in establishing the mammalian complement system had occurred by the time of the teleost/mammalian divergence around 500 million years ago (MYA). Members of most complement gene families are also present in ascidians, although they do not show a one-to-one correspondence to their counterparts in higher vertebrates, indicating that the gene duplications of each gene family occurred independently in vertebrates and ascidians. The C3 and factor B genes, but probably not the other complement genes, are present in the genome of the cnidaria and some protostomes, indicating that the origin of the central part of the complement system was established more than 1,000 MYA.

• Long S et al.
• Identification and expression analysis of cDNAs encoding channel catfish type I interferons.
• Fish Shellfish Immunol. 2006; 21: 42-59
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• Previously a cDNA encoding a putative interferon gene, designated CF IFN-1, was identified from a catfish EST library. However, its constitutive expression, absence of a signal peptide, and apparently low level of biological activity suggested that this cDNA likely encoded an expressed pseudogene. Since Southern blot analysis suggested the presence of two to three IFN genes, additional cDNAs were generated from catfish fibroblast and lymphoid cell lines using primers designed to conserved regions of zebrafish and catfish interferon. Using this approach, three novel CF IFN genes, two of which likely encode functional interferon molecules, were identified. At the amino acid level, similarity among CF IFNs ranged from 71% to 82%, whereas similarity to other fish IFNs ranged from 15% to 35%. Although CF IFN-3, like CF IFN-1, lacks a signal peptide, CF IFN-2 and -4 appear to encode full-length, signal sequence-bearing genes. Consistent with their putative identification as functional genes, CF IFN-2 and -4 were not expressed in unstimulated cell lines, and CF IFN-2 was rapidly upregulated in CCO cells in response to virus infection or treatment with dsRNA. Moreover, as with salmon, fugu, and zebrafish interferon genes, CF IFN-1 contained four introns whose locations were conserved not only with respect to other fish IFNs, but also with respect to mammalian IFN-lambda. While it is likely that CF IFNs represent Type I IFNs, several characteristics preclude assigning these cytokines to any particular subfamily.

• Krushna Padhi B, Akimenko MA, Ekker M
• Independent expansion of the keratin gene family in teleostean fish and mammals: an insight from phylogenetic analysis and radiation hybrid mapping of keratin genes in zebrafish.
• Gene. 2006; 368: 37-45
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• The sequence and chromosomal distribution of keratin genes of zebrafish were compared with that of other fishes and mammals to provide an insight into the evolution of this gene family in vertebrates. By comparative sequence analysis and radiation hybrid mapping, we identified 16 type I and 7 type II keratin genes in the zebrafish genome. This contrasts with mammals, where type I and type II keratin genes are similar in number. The keratin genes are scattered in the fish genome, contrasting with the two clusters of keratin genes in mammalian genomes. Compared to genes from two species of pufferfish, the zebrafish type I keratin genes underwent an expansion by independent tandem duplications. Expression profiles based on EST counts suggest that some of the tandemly duplicated type I keratin genes from zebrafish either underwent sub-functionalization or acquired new expression domains. The chromosomal arrangement of keratins 8, keratin18, and a second type II keratin, as a cluster of three genes, has remained conserved in vertebrate evolution, except for duplication of the three-gene cluster in some teleosts. This contrasts with other members of the keratin gene family, which diverged independently between fish and mammals.

• Kedinger V, Alpy F, Tomasetto C, Thisse C, Thisse B, Rio MC
• Spatial and temporal distribution of the traf4 genes during zebrafish development.
• Gene Expr Patterns. 2005; 5: 545-52
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• The tumor necrosis factor-associated factor 4 (TRAF4) is a particular member of the TRAF protein family since it is not involved in the Tumor Necrosis Factor (TNF) and Interleukin-1 (IL-1) signaling pathways. In the present study, we cloned two zebrafish orthologs of the human traf4, traf4a and traf4b, which are the first TRAFs described in zebrafish. During embryogenesis, traf4b expression is present in a weak ubiquitous manner. In contrast, traf4a exhibits a highly specific expression pattern in the sensorial and neural cells, and the somites of embryos. This gene is tightly regulated during embryogenesis. Together, our data show that traf4 is conserved during evolution, and traf4a is the zebrafish ortholog of traf4.

• Volff JN
• Genome evolution and biodiversity in teleost fish.
• Heredity (Edinb). 2005; 94: 280-94
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• Teleost fish, which roughly make up half of the extant vertebrate species, exhibit an amazing level of biodiversity affecting their morphology, ecology and behaviour as well as many other aspects of their biology. This huge variability makes fish extremely attractive for the study of many biological questions, particularly of those related to evolution. New insights gained from different teleost species and sequencing projects have recently revealed several peculiar features of fish genomes that might have played a role in fish evolution and speciation. There is now substantial evidence that a round of tetraploidization/rediploidization has taken place during the early evolution of the ray-finned fish lineage, and that hundreds of duplicate pairs generated by this event have been maintained over hundreds of millions of years of evolution. Differential loss or subfunction partitioning of such gene duplicates might have been involved in the generation of fish variability. In contrast to mammalian genomes, teleost genomes also contain multiple families of active transposable elements, which might have played a role in speciation by affecting hybrid sterility and viability. Finally, the amazing diversity of sex determination systems and the plasticity of sex chromosomes observed in teleost might have been involved in both pre- and postmating reproductive isolation. Comparison of data generated by current and future genome projects as well as complementary studies in other species will allow one to approach the molecular and evolutionary mechanisms underlying genome diversity in fish, and will certainly significantly contribute to our understanding of gene evolution and function in humans and other vertebrates.

• Zhang DC, Shao YQ, Huang YQ, Jiang SG
• Cloning, characterization and expression analysis of interleukin-10 from the zebrafish (Danio rerion).
• J Biochem Mol Biol. 2005; 38: 571-6
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• Cytokines are proteins produced by many different cells of the immune system and play a significant role in initiating and regulating the inflammatory process. In this research, an important cytokine, interleukin-10 (IL-10) gene, has been identified and characterized from zebrafish (Danio rerio) genome database. Zebrafish IL-10 is located within a 2690 bp fragment and contains five exons and four introns, sharing the same organization with mammalian IL-10 genes. An open reading frame of 543 bp was found to encode a putative 180 amino acid protein with a signal peptide of 22 amino acids, which shares 29.7-80.9 % homology with amino acid sequences of other known IL-10. The signature motif of IL-10 is also conserved in zebrafish IL-10. The predicted transcript was finally confirmed by sequencing of cDNA clones. Multi-tissue reverse transcriptase PCR (RT-PCR) was performed to examine the tissue distribution and expression regulation of this gene in seven organs of normal and lipopolysaccharide (LPS) stimulation zebrafish. The results demonstrated that this gene was expressed slightly in normal kidney, gill and gut, no expression was detected in other four tissues. The expression was clearly upregulated after LPS stimulation. Using the ideal zebrafish model, further study of IL-10 characterization and function may provide insight on the understanding of the innate immune system.

• Gloriam DE, Bjarnadottir TK, Yan YL, Postlethwait JH, Schioth HB, Fredriksson R
• The repertoire of trace amine G-protein-coupled receptors: large expansion in zebrafish.
• Mol Phylogenet Evol. 2005; 35: 470-82
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• Trace amines, such as tyramine, beta-phenylethylamine, tryptamine, and octopamine, are present in trace levels in nervous systems and bind a specific family of G-protein-coupled receptors (GPCR), but the function or origin of this system is not well understood. We searched the genomes of several eukaryotic species for receptors similar to the mammalian trace amine (TA) receptor subfamily. We identified 18 new receptors in rodents that are orthologous to the previously known TA-receptors. Remarkably, we found 57 receptors (and 40 pseudogenes) of this type in the zebrafish (Danio rerio), while fugu (Takifugu rubripes) had only eight receptors (and seven pseudogenes). We mapped 47 of the zebrafish TA-receptors on chromosomes using radiation hybrid panels and meiotic mapping. The results, together with the degree of conservation and phylogenetic relationships displayed among the zebrafish receptors suggest that the family arose through several different mechanisms involving tetraploidization, block duplications, and local duplication events. Interestingly, these vertebrate TA-receptors do not show a close evolutionary relationship to the invertebrate TA-binding receptors in fruitfly (Drosophila melanogaster), indicating that the ability to bind TA have evolved at least twice in animal evolution. We collected in total over 100 vertebrate TA-receptor sequences, and our phylogenetic analysis shows that several TA-receptors have evolved rapidly with remarkable species variation and that the common ancestor of vertebrate TA-receptors arose before the split of the ray-finned and lobe-finned fishes. The evolutionary history of the TA-receptors is more complex than for most other GPCR families and here we suggest a mechanism by which they may have arisen.

• Phelan PE, Mellon MT, Kim CH
• Functional characterization of full-length TLR3, IRAK-4, and TRAF6 in zebrafish (Danio rerio).
• Mol Immunol. 2005; 42: 1057-71
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• Recently, the zebrafish, Danio rerio, has been recognized as a useful model for infectious disease and immunity. The Toll-like receptor (TLR) family is an evolutionarily conserved component of the innate immune system that responds to specific pathogen-associated molecular patterns (PAMPs) during an infection. This study reports the identification and characterization of a full-length orthologue of mammalian TLR3, and the key TLR pathway signaling molecules IRAK-4 and TRAF6 in the zebrafish. Sequence analysis of zebrafish TLR3 (zfTLR3), IRAK-4 (zfIRAK-4), and TRAF6 (zfTRAF6) revealed conserved domains shared with insect and mammalian genes. Quantitative real-time PCR showed that all three genes are expressed in a variety of adult tissues and during embryonic development. In in situ hybridization, we showed that zfTLR3, zfIRAK-4, and zfTRAF6 are present in distinct regions of the developing brain at 22hpf and that zfTRAF6 was observed in the developing medial neural tube. Overexpression of zfIRAK-4, zfTRAF6, or a mutant zfTLR3 construct was able to stimulate NF-kappaB activation in ZFL cells as measured by a cotransfected NF-kappaB-luciferase reporter plasmid. Messenger RNA expression profiles of each gene in zebrafish embryos and adults were examined by quantitative real-time PCR following infection with snakehead rhabdovirus (SHRV) or Edwardsiella tarda. Following exposure to SHRV, only zfTLR3 and zfTRAF6 mRNA transcripts were upregulated. Interestingly, exposure of fish to E. tarda resulted in an unexpected increase in mRNA expression of zfTLR3, as well as the anticipated upregulation of zfIRAK-4 and zfTRAF6 mRNA transcripts. These results demonstrate that zebrafish possess conserved TLR-signaling pathways, further emphasizing the utility of the zebrafish as a model for vertebrate immunology.

• Irwin DM, Wong K
• Evolution of new hormone function: loss and gain of a receptor.
• J Hered. 2005; 96: 205-11
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• The vertebrate proglucagon gene encodes three glucagon-like sequences (glucagon, glucagon-like peptide-1 [GLP-1], and glucagon-like peptide 2 [GLP-2]) that have distinct functions in regulating metabolism in mammals. In contrast, glucagon and GLP-1 have similar physiological actions in fish, that of mammalian glucagon. We have identified sequences similar to receptors for proglucagon-derived peptides from the genomes of two fish (pufferfish and zebrafish), a frog (Xenopus tropicalis), and a bird (chicken). Phylogenetic analysis of the receptor sequences suggested an explanation for the divergent function of GLP-1 in fish and mammals. The phylogeny of our predicted and characterized receptors for proglucagon-derived peptides demonstrate that receptors for glucagon, GLP-1, and GLP-2 have an origin before the divergence of fish and mammals; however, fish have lost the gene encoding the GLP-1 class of receptors, and likely the incretin action of GLP-1. Receptors that bind GLP-1, but yield glucagon-like action, have been characterized in goldfish and zebrafish, and these sequences are most closely related to glucagon receptors. Both pufferfish and zebrafish have a second glucagon receptor-like gene that is most closely related to the characterized goldfish glucagon receptor. The phylogeny of glucagon receptor-like genes in fish indicates that a duplication of the glucagon receptor gene occurred on the ancestral fish lineage, and could explain the shared action of glucagon and GLP-1. We suggest that the binding specificity of one of the duplicated glucagon receptors has diverged, yielding receptors for GLP-1 and glucagon, but that ancestral downstream signaling has been maintained, resulting in both receptors retaining glucagon-stimulated downstream effects.

• Premzl M, Gready JE, Jermiin LS, Simonic T, Marshall Graves JA
• Evolution of vertebrate genes related to prion and Shadoo proteins--clues from comparative genomic analysis.
• Mol Biol Evol. 2004; 21: 2210-31
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• Recent findings of new genes in fish related to the prion protein (PrP) gene PRNP, including our recent report of SPRN coding for Shadoo (Sho) protein found also in mammals, raise issues of their function and evolution. Here we report additional novel fish genes found in public databases, including a duplicated SPRN gene, SPRNB, in Fugu, Tetraodon, carp, and zebrafish encoding the Sho2 protein, and we use comparative genomic analysis to analyze the evolutionary relationships and to infer evolutionary trajectories of the complete data set. Phylogenetic footprinting performed on aligned human, mouse, and Fugu SPRN genes to define candidate regulatory promoter regions, detected 16 conserved motifs, three of which are known transcription factor-binding sites for a receptor and transcription factors specific to or associated with expression in brain. This result and other homology-based (VISTA global genomic alignment; protein sequence alignment and phylogenetics) and context-dependent (genomic context; relative gene order and orientation) criteria indicate fish and mammalian SPRN genes are orthologous and suggest a strongly conserved basic function in brain. Whereas tetrapod PRNPs share context with the analogous stPrP-2-coding gene in fish, their sequences are diverged, suggesting that the tetrapod and fish genes are likely to have significantly different functions. Phylogenetic analysis predicts the SPRN/SPRNB duplication occurred before divergence of fish from tetrapods, whereas that of stPrP-1 and stPrP-2 occurred in fish. Whereas Sho appears to have a conserved function in vertebrate brain, PrP seems to have an adaptive role fine-tuned in a lineage-specific fashion. An evolutionary model consistent with our findings and literature knowledge is proposed that has an ancestral prevertebrate SPRN-like gene leading to all vertebrate PrP-related and Sho-related genes. This provides a new framework for exploring the evolution of this unusual family of proteins and for searching for members in other fish branches and intermediate vertebrate groups.

• Damiano JS, Oliveira V, Welsh K, Reed JC
• Heterotypic interactions among NACHT domains: implications for regulation of innate immune responses.
• Biochem J. 2004; 381: 213-9
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• Proteins of the NACHT [NAIP (neuronal apoptosis inhibitory protein), CIITA (MHC class II transcription activator), HET-E (incompatibility locus protein from Podospora anserina) and TP1 (telomerase-associated protein)] family may serve as critical pathogen-sensing and signal-transducing molecules within the innate immune system. In the present paper, we show that CLAN [CARD (caspase-recruitment domain), LRR (leucine-rich repeat) and NACHT domain-containing protein], a NACHT-containing protein originally demonstrated to bind and activate pro-caspase 1, is also capable of influencing the functions of other members of the NACHT family. Through heterotypic NACHT-domain interactions, CLAN was found to associate with Nod1, Nod2 and NAC [nucleotide-binding domain and CARD-containing protein; NALP1 (NACHT, LRR and PYRIN protein 1)] when co-expressed in HEK-293T (human embryonic kidney) cells. NF-kappaB (nuclear factor kappaB) reporter assays demonstrated that co-expression of either full-length CLAN or the NACHT domain of CLAN significantly inhibited NF-kappaB activation induced by Nod1 or Nod2 overexpression. In addition, co-expression of CLAN or the NACHT domain of CLAN with Nod1 or Nod2 inhibited the ability of these proteins to generate active IL-1beta (interleukin 1beta) through their association with pro-caspase 1. The NACHT domain of CLAN was demonstrated by co-immunoprecipitation experiments to bind all NACHT domains that were tested, including the NACHT domains from CLAN itself, Nod1, Nod2, cryopyrin, NAC, PAN2 [PAAD [pyrin, AIM (absent-in-melanoma), ASC (apoptosis-associated speck-like protein containing a CARD) and death-domain-like]- and NACHT-containing protein] and NAIP (neuronal apoptosis inhibitory protein). Finally, monocyte-expressed CLAN was found to associate with Nod2 following exposure to bacterial peptidoglycan, implying a regulatory role for interaction of these NACHT proteins in the innate immune response. These studies suggest that by mediating hetero-oligomerization, NACHT domains provide a means by which various NACHT-containing proteins may interact, creating protein-interaction networks that potentially modulate immune responses to invading pathogens.

• Meijer AH et al.
• Expression analysis of the Toll-like receptor and TIR domain adaptor families of zebrafish.
• Mol Immunol. 2004; 40: 773-83
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• The zebrafish genomic sequence database was analysed for the presence of genes encoding members of the Toll-like receptors (TLR) and interleukin receptors (IL-R) and associated adaptor proteins containing a TIR domain. The resulting predictions show the presence of one or more counterparts for the human TLR1, TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, IL-1R and IL-18R genes and one copy of the adaptor genes MyD88, MAL, TRIF and SARM. In contrast to data for the pufferfish Fugu rubripes, zebrafish has two genes that are highly similar to human TLR4. In addition, one fish-specific TLR group can be distinguished that is closely related to the Drosophila melanogaster Toll-9 gene. The sequence of cloned cDNAs for TLR4, TLR2 and MyD88 show the same intron-exon organisation as in the human counterparts. Expression analysis using reverse transcriptase-PCR (RT-PCR) shows that 17 of the predicted zebrafish TLR genes and all the genes encoding adaptor proteins are expressed in the adult stage. A subset of the TLR genes are expressed at higher levels in fish infected with the pathogen Mycobacterium marinum. The induced genes include the homologues of the human TLR1 and TLR2 genes, whose functions are associated with mycobacterial infections, underscoring the suitability of zebrafish as a model for analysis of the vertebrate innate immune system.

• Dildrop R, Ruther U
• Organization of Iroquois genes in fish.
• Dev Genes Evol. 2004; 214: 267-76
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• In mammals, a total of six iroquois ( Irx) genes exist, which are organized into two clusters. Here we report on the organization of all iroquois genes present in fish, using zebrafish ( Danio rerio) and pufferfish ( Fugu rubripes and Tetraodon nigroviridis) as examples. A total of 10 Irx genes were found in pufferfish, and 11 in zebrafish; all but one of these genes are organized into clusters (four clusters plus one isolated gene locus). The "extra" fish clusters result from chromosome duplication in the fish lineage, after its divergence from tetrapod vertebrates. Two of the four fish clusters are highly conserved to the ones in mammals, with regard to similarity of genes and cluster architecture. Irx genes within the other two clusters have diverged in sequence and cluster organization, suggesting functional divergence. These results will allow us to use the zebrafish system for functional and comparative studies of iroquois genes in vertebrate development.

• Oshiumi H, Tsujita T, Shida K, Matsumoto M, Ikeo K, Seya T
• Prediction of the prototype of the human Toll-like receptor gene family from the pufferfish, Fugu rubripes, genome.
• Immunogenetics. 2003; 54: 791-800
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• The insect Toll family of proteins and their mammalian counterparts seemingly shared one common ancestor and evolved independently. Here we demonstrated that the prototype of the mammalian-type (M-type) Toll family is shared by the fish and humans. According to the draft of the pufferfish Fugu genome project, the signature Toll-IL-1 receptor homology domain (TIR domain) has been conserved during evolution. FuguTLR2, 3, 5, 7, 8 and 9 members correspond structurally to respective mammalian TLRs. One Fugu TLR showed equally high amino acid identity to human TLR1, 6 and 10, and we named it FuguTLR1. Fugu rubripes has genes for TLR21 and 22, which are unique to fish. One possible interpretation of these findings is that TLR1, 2, 3, 4, 5, 7, 8, 9, 21 and 22 existed in the ancestral genome common to fish and mammals, and that TLR4 was lost in the fish lineage, while TLR21 and 22 were lost in the mammalian lineage. Strikingly, a solitary ascidian, Halocynthia roretzi, has only a few Toll-like proteins, which, like Caenorhabditis elegans Toll, represent primitive ones before the expansion of the Toll family. Therefore, the expansion of TLR genes should have occurred earlier than fish, but not C. intestinalis, separated evolutionarily from mammals. These results infer that the appearance of the M-type innate system was completed before or concomitant with the appearance of acquired immunity. We interpret the present data to mean that the differences of TLRs identified in this study between fishes and humans may be rather peripheral, partially due to selection pressure exerted by pathogens in distinct environments.

• Traver D et al.
• The zebrafish as a model organism to study development of the immune system.
• Adv Immunol. 2003; 81: 253-330

• Engelsma MY et al.
• Neuroendocrine-immune interactions in fish: a role for interleukin-1.
• Vet Immunol Immunopathol. 2002; 87: 467-79
• Display abstract

• Bi-directional communication between the hypothalamus-pituitary-adrenal (HPA)-axis and the sympathetic nervous system with the immune system is crucial to ensure homeostasis. Shared use of ligands and especially receptors forms a key component of this bi-directional interaction. Glucocorticoids (GC), the major end products of the HPA-axis differentially modulate immune function. Cytokines, especially interleukin-1 (IL-1), tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), ensure immune signalling to the neuroendocrine system. In addition, hormones from leukocyte origin such as corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and beta-endorphin, as well as centrally synthesised and secreted cytokines, contribute to the communication network. In teleost fish cortisol is the major product of the hypothalamus-pituitary-interrenal (HPI)-axis which is the teleost equivalent of the HPA-axis. Moderate and substantial increases in cortisol during stressful circumstances negatively affect B-lymphocytes, whereas rescue of neutrophilic granulocytes may support innate immunity. Recent elucidation of lower vertebrate cytokine sequences has facilitated research into neuroendocrine-immune interactions in teleosts and the first evidence for a significant function of interleukin-1 in the bi-directional communication is discussed.

• Kortschak RD, Tamme R, Lardelli M
• Evolutionary analysis of vertebrate Notch genes.
• Dev Genes Evol. 2001; 211: 350-4
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• We have conducted an evolutionary analysis of Notch genes of the vertebrates Danio rerio and Mus musculus to examine the expansion and diversification of the Notch family during vertebrate evolution. The existence of multiple Notch genes in vertebrate genomes suggests that the increase in Notch signaling pathways may be necessary for the additional complexity observed in the vertebrate body plan. However, orthology relationships within the vertebrate Notch family indicate that biological functions are not fixed within orthologous groups. Phylogenetic reconstruction of the vertebrate Notch family suggests that the zebrafish notch1a and 1b genes resulted from a duplication occurring around the time of the teleost/mammalian divergence. There is also evidence that the mouse Notch4 gene is the result of a rapid divergence from a Notch3-like gene. Investigation of the ankyrin repeat region sequences showed there to be little evidence for gene conversion events between repeat units. However, relationships between repeats 2-5 suggest that these repeats are the result of a tandem duplication of a dual repeat unit. Selective pressure on maintenance of ankyrin repeat sequences indicated by relationships between the repeats suggests that specific repeats are responsible for particular biological activities, a finding consistent with mutational studies of the Caenorhabditis elegans gene glp-1. Sequence similarities between the ankyrin repeats and the region immediately C-terminal of the repeats further suggests that this region may be involved in the modulation of ankyrin repeat function.