Corine P. Kruiswijk

Molecular evolution of CXC chemokines: extant CXC chemokines originate from the CNS

The mammalian CXC chemokine system comprises 16 ligands and six receptors, and its actions stretch well beyond the immune system. Recent elucidation of the pufferfish genome, a representative of an evolutionary ancient vertebrate class, has enabled analysis of the mammalian CXC chemokine system in a phylogenetic context. Comparison of the phylogenies of vertebrate CXC chemokines revealed that fish and mammals have found different solutions to similar problems, grafted on the same basic structural motif. Phylogenetic analyses showed that the large, highly redundant CXC chemokine family is a very recent phenomenon that is exclusive to higher vertebrates. Moreover, its ancestral role is found within the central nervous system and not within the immune system.

A novel functional class I lineage in zebrafish (Danio rerio), carp (Cyprinus carpio), and large barbus (Barbus intermedius) showing an unusual conservation of the peptide binding domains.

Species from all major jawed vertebrate taxa possess linked polymorphic class I and II genes located in an MHC. The bony fish are exceptional with class I and II genes located on different linkage groups. Zebrafish (Danio rerio), common carp (Cyprinus carpio), and barbus (Barbus intermedius) represent highly divergent cyprinid genera. The genera Danio and Cyprinus diverged 50 million years ago, while Cyprinus and Barbus separated 30 million years ago. In this study, we report the first complete protein-coding class I ZE lineage cDNA sequences with high similarity between the three cyprinid species. Two unique complete protein-coding cDNA sequences were isolated in zebrafish, Dare-ZE*0101 and Dare-ZE*0102, one in common carp, Cyca-ZE*0101, and six in barbus, Bain-ZE*0101, Bain-ZE*0102, Bain-ZE*0201, Bain-ZE*0301, Bain-ZE*0401, and Bain-ZE*0402. Deduced amino acid sequences indicate that these sequences encode bonafide class I proteins. In addition, the presence of conserved potential peptide anchoring residues, exon-intron organization, ubiquitous expression, and polymorphism generated by positive selection on putative peptide binding residues support a classical nature of class I ZE lineage genes. Phylogenetic analyses revealed clustering of the ZE lineage clade with nonclassical cyprinid class I Z lineage clade away from classical cyprinid class I genes, suggesting a common ancestor of these nonclassical genes as observed for mammalian class I genes. Data strongly support the classical nature of these ZE lineage genes that evolved in a trans-species fashion with lineages being maintained for up to 100 million years as estimated by divergence time calculations.

Multiple and highly divergent IL-11 genes in teleost fish

Interleukin-11 (IL-11) is a key cytokine in the regulation of proliferation and differentiation of hematopoietic progenitors and is also involved in bone formation, adipogenesis, and protection of mucosal epithelia. Despite this prominent role in diverse physiological processes, IL-11 has been described in only four mammalian species, and recently, in rainbow trout (Oncorhynchus mykiss). Here we report the presence of IL-11 in common carp (Cyprinus carpio), a bony fish species related to zebrafish. IL-11 is expressed in most carp organs and tissues. In vitro expression of IL-11 in cultured macrophages is enhanced by stimulation with lipopolysaccharide and is markedly inhibited by cortisol. A detailed and systematic scan of several fish genome databases confirms that IL-11 is present in all fish, but also reveals the presence of a second, substantially different IL-11 gene in the genomes of phylogenetically distant fish species. We designated both fish paralogues IL-11a and IL-11b. Although sequence identity between fish IL-11a and IL-11b peptides is low, the conservation of their gene structures supplemented by phylogenetic analyses clearly illustrate the orthology of both IL-11a and IL-11b genes of fish with mammalian IL-11. The presence of IL-11 genes in fish demonstrates its importance throughout vertebrate evolution, although the presence of duplicate and divergent IL-11 genes differs from the single IL-11 gene that exists in mammals.

Major histocompatibility genes in the Lake Tana African large barb species flock: evidence for complete partitioning of class II B, but not class I, genes among different species

The 16 African ‘large’ barb fish species of Lake Tana inhabit different ecological niches, exploit different food webs and have different temporal and spatial spawning patterns within the lake. This unique fish species flock is thought to be the result of adaptive radiation within the past 5 million years. Previous analyses of major histocompatibility class II B exon 2 sequences in four Lake Tana African large barb species revealed that these sequences are indeed under selection. No sharing of class II B alleles was observed among the four Lake Tana African large barb species. In this study we analysed the class II B exon 2 sequences of seven additional Lake Tana African large barb species and African large barbs from the Blue Nile and its tributaries. In addition, the presence and variability of major histocompatibility complex class I UA exon 3 sequences in six Lake Tana and Blue Nile African large barb species was analysed. Phylogenetic lineages are maintained by purifying or neutral selection on non-peptide binding regions. Class II B intron 1 and exon 2 sequences were not shared among the different Lake Tana African large barb species or with the riverine barb species. In contrast, identical class I UA exon 3 sequences were found both in the lacustrine and riverine barb species. Our analyses demonstrate complete partitioning of class II B alleles among Lake Tana African large barb species. In contrast, class I alleles remain for the large part shared among species. These different modes of evolution probably reflect the unlinked nature of major histocompatibility genes in teleost fishes.

Response to Shields: Molecular evolution of CXC chemokines and receptors

The letter by Denis Shields [1] comments on our article on the molecular evolution of CXC chemokines and their receptors [2], in which we hypothesized that the chemokines originate from the central nervous system (CNS). Shield’s comments hinged mainly on the evolutionary analyses. Clearly, inferences drawn from phylogenetic analyses do have their limitations. In the initial stages of the analyses, we constructed phylogenetic trees using an outgroup for both receptor and ligands. Although in general outgroups are chosen arbitrarily, we constructed a CXC receptor (CXCR) tree with CC chemokine receptors (CCRs) as an outgroup. The topology did not change from what was presented, except for CXCR6, which was located in the CCR cluster, close to the starting point of cluster formation but with low bootstrap values. In general, the tips of a phylogenetic tree are well resolved, whereas the deeper nodes are poorly resolved. ‘Supertrees’, which in theory could combine different types of information, and not merely DNA or protein sequence information, might be an alternative for studying long evolutionary histories [3]. We adopted the philosophy of a ‘supertree’ approach by substantiating our hypothesis using not only phylogenetic analyses but also other information, such as protein function, several CXC ligands (CXCL) sharing the same receptor, the presence of the ELR (glutamic acid, leucine, arginine) motif, and chromosomal localizations. Shields also argues that the absence of CXCR4 in Ciona favours a primordial immunological function of the CXC family. However, in this respect, the presence of CXCR4 in jawless fish is more important. Kuroda et al. [4] sequenced 9312 cDNA clones from a lamprey (Petromyzon marinus). The serendipitous finding of only CXCR4 could be a testimony to its ancestral status. The lamprey CXCR4 receptors cluster, with a high bootstrap value, with all other CXCR4 found in jawed fish, amphibia, birds and mammals. In addition, the architecture and sophistication of the CNS of Ciona differ markedly compared with that found in other more advanced chordates [5]. Although using a small number of amino acids to construct a phylogeny can lead to incorrect topologies, this can be overcome by the bootstrap test [6], which we used extensively to identify statistically reliable orthologues. The use of complete sequences is preferred to detect true orthologues, although when only incomplete sequences are available, the phylogenetic analyses can be performed in two ways, using pair-wise deletion or complete deletion of missing information. We performed both, and the results were essentially identical topologies with only minor deviations, notably the position of the Xenopus CXCR3 and rainbow trout CXCR. Phylogenetic trees are a hypothetical model for the evolutionary history of a gene family. A gene conversion event might result in two paralogous genes clustering closer together in trees than in reality. Gene conversions occur relatively frequently; Shields [7] observed that 45% of a control group of clustered genes showed evidence of gene conversion. However, this should not preclude phylogenetic analyses; we should merely be aware of the possibility of gene conversion imposing a bias on the analyses. The gene conversion events between the clustered CXCR1 and CXCR2 sequences analysed by Shields [7] using human, rabbit and rat sequences probably occurred between ,90 and 110 million years ago [8]. The evolutionary scale on which these gene conversions could bias the phylogenetic tree is therefore limited to the clustering of CXCR1 and CXCR2 in mammals. We believe that putative gene conversion events do not interfere with our conclusions and hypotheses derived from phylogenetic analyses on a larger, vertebrate-wide scale that comprises more CXCRs. Shields also touches upon the controversy surrounding genome duplication. Some studies favour whole-genome duplications, whereas others point to accumulated duplication of blocks or single chromosomes. Most data supporting whole genome duplications are derived from analyses of either the major histocompatibility complex (MHC) [9] or the Hox [10] gene clusters. This issue is still unresolved. Following either mode of duplication, it is apparent that extensive reshuffling and loss of genes or gene regions has occurred. This obscures the reconstruction of events occurring during the ‘birth’ of genomes [11]. Our hypotheses do not refute either the whole-genome duplication or the block duplication hypothesis. Regardless of the pitfalls associated with phylogenetic analyses, this approach has the potential to unravel the evolutionary history of the CXCL/CXCR multigene families. Our opinion, based on a combination of these analyses and additional data, is an attempt to set these gene families in a compelling evolutionary perspective. Corresponding author: B.M. Lidy Verburg-van Kemenade (lidy.vankemenade@ wur.nl). Update TRENDS in Immunology Vol.24 No.7 July 2003 356