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The immune system employs soluble effectors to shape luminal spaces. Antibodies are soluble molecules that effect immunological responses, including neutralization, opsonization, antibody-dependent cytotoxicity and complement activation. These molecules are comprised of immunoglobulin (Ig) domains. The N-terminal Ig domains recognize antigen, and the C-terminal domains facilitate their elimination through phagocytosis (opsonization). A less-recognized function mediated by the C-terminal Ig domains of the IgG class of antibodies (Fc region) involves the formation of multiple low-affinity bonds with the mucus matrix. This association anchors the antibody molecule to the matrix to entrap potential pathogens. Even though invertebrates are not known to have antibodies, protochordates have a class of secreted molecules containing Ig domains that can bind bacteria and potentially serve a similar purpose. The VCBPs (V region-containing chitin-binding proteins) possess a C-terminal chitin-binding domain that helps tether them to chitin-rich mucus gels, mimicking the IgG-mediated Fc trapping of microbes in mucus. The broad functional similarity of these structurally divergent, Ig-containing, secreted effectors makes a case for a unique form of convergent evolution within chordates. This opinion essay highlights emerging evidence that divergent secreted immune effectors with Ig-like domains evolved to manage immune recognition at mucosal surfaces in strikingly similar ways. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.

Multiple novel immunoglobulin-like transcripts (NILTs) have been identified from salmon, trout, and carp. NILTs typically encode activating or inhibitory transmembrane receptors with extracellular immunoglobulin (Ig) domains. Although predicted to provide immune recognition in ray-finned fish, we currently lack a definitive framework of NILT diversity, thereby limiting our predictions for their evolutionary origin and function. In order to better understand the diversity of NILTs and their possible roles in immune function, we identified five NILT loci in the Atlantic salmon (Salmo salar) genome, defined 86 NILT Ig domains within a 3-Mbp region of zebrafish (Danio rerio) chromosome 1, and described 41 NILT Ig domains as part of an alternative haplotype for this same genomic region. We then identified transcripts encoded by 43 different NILT genes which reflect an unprecedented diversity of Ig domain sequences and combinations for a family of non-recombining receptors within a single species. Zebrafish NILTs include a sole putative activating receptor but extensive inhibitory and secreted forms as well as membrane-bound forms with no known signaling motifs. These results reveal a higher level of genetic complexity, interindividual variation, and sequence diversity for NILTs than previously described, suggesting that this gene family likely plays multiple roles in host immunity., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The gut microbiome of animals consists of diverse microorganisms that include both prokaryotes and eukaryotes. Complex interactions occur among these inhabitants, as well as with the immune system of the host, and profoundly influence the overall health of both the host and its microbial symbionts. Despite the enormous importance for the host to regulate its gut microbiome, the extent to which animals generate immune-related molecules with the capacity to directly influence polymicrobial interactions remains unclear. The urochordate, Ciona robusta , is a model organism that has been adapted to experimental studies of host/microbiome interactions. Ciona variable-region containing chitin-binding proteins (VCBPs) are innate immune effectors, composed of immunoglobulin (Ig) variable regions and a chitin-binding domain (CBD) and are expressed in high abundance in the gut. It was previously shown that VCBP-C binds bacteria and influences both phagocytosis by granular amoebocytes and biofilm formation via its Ig domains. We show here that the CBD of VCBP-C independently recognizes chitin molecules present in the cell walls, sporangia (spore-forming bodies), and spores of a diverse set of filamentous fungi isolated from the gut of Ciona . To our knowledge, this is the first description of a secreted Ig-containing immune molecule with the capacity to directly promote transkingdom interactions through simultaneous binding by independent structural domains and could have broad implications in modulating the establishment, succession, and homeostasis of gut microbiomes.

The gastrointestinal tract of Ciona intestinalis , a solitary tunicate that siphon-filters water, shares similarities with its mammalian counterpart. The Ciona gut exhibits other features that are unique to protochordates, including certain immune molecules, and other characteristics, e.g. chitin-rich mucus, which appears to be more widespread than considered previously. Exposure of Ciona to dextran sulphate sodium (DSS) induces a colitis-like phenotype similar to that seen in other systems, and is characterized by alteration of epithelial morphology and infiltration of blood cells into lamina propria-like regions. DSS treatment also influences the production and localization of a secreted immune molecule shown previously to co-localize to chitin-rich mucus in the gut. Resistance to DSS is enhanced by exposure to exogenous chitin microparticles, suggesting that endogenous chitin is critical to barrier integrity. Protochordates, such as Ciona , retain basic characteristics found in other more advanced chordates and can inform us of uniquely conserved signals shaping host-microbiota interactions in the absence of adaptive immunity. These simpler model systems may also reveal factors and processes that modulate recovery from colitis, the role gut microbiota play in the onset of the disease, and the rules that help govern the reestablishment and maintenance of gut homeostasis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The resolution of the gar genome affords an opportunity to examine the diversification and functional specialization of immune effector molecules at a distant and potentially informative point in phylogenetic development. Although innate immunity is effected by a particularly large number of different families of molecules, the focus here is to provide detailed characterization of several families of innate receptors that are encoded in large multigene families, for which orthologous forms can be identified in other species of bony fish but not in other vertebrate groups as well as those for which orthologs are present in other vertebrate species. The results indicate that although teleost fish and the gar, as a holostean reference species, share gene families thought previously to be restricted to the teleost fish, the manner in which the members of the multigene families of innate immune receptors have undergone diversification is different in these two major phylogenetic radiations. It appears that both the total genome duplication and different patterns of genetic selection have influenced the derivation and stabilization of innate immune genes in a substantial manner during the course of vertebrate evolution., (© 2017 Wiley Periodicals, Inc.)

Bony fish encode multiple multi-gene families of membrane receptors that are comprised of immunoglobulin (Ig) domains and are predicted to function in innate immunity. One of these families, the diverse immunoglobulin (Ig) domain-containing protein (DICP) genes, maps to three chromosomal loci in zebrafish. Most DICPs possess one or two Ig ectodomains and include membrane-bound and secreted forms. Membrane-bound DICPs include putative inhibitory and activating receptors. Recombinant DICP Ig domains bind lipids with varying specificity, a characteristic shared with mammalian CD300 and TREM family members. Numerous DICP transcripts amplified from different lines of zebrafish did not match the zebrafish reference genome sequence suggesting polymorphic and haplotypic variation. The expression of DICPs in three different lines of zebrafish has been characterized employing PCR-based strategies. Certain DICPs exhibit restricted expression in adult tissues whereas others are expressed ubiquitously. Transcripts of a subset of DICPs can be detected during embryonic development suggesting roles in embryonic immunity or other developmental processes. Transcripts representing 11 previously uncharacterized DICP sequences were identified. The assignment of two of these sequences to an unplaced genomic scaffold resulted in the identification of an alternative DICP haplotype that is linked to a MHC class I Z lineage haplotype on zebrafish chromosome 3. The linkage of DICP and MHC class I genes also is observable in the genomes of the related grass carp (Ctenopharyngodon idellus) and common carp (Cyprinus carpio) suggesting that this is a shared character with the last common Cyprinidae ancestor.

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences.

Protochordate variable region-containing chitin-binding proteins (VCBPs) consist of immunoglobulin-type V domains and a chitin-binding domain (CBD). VCBP V domains facilitate phagocytosis of bacteria by granulocytic amoebocytes; the function of the CBD is not understood. Here we show that the gut mucosa of Ciona intestinalis contains an extensive matrix of chitin fibrils to which VCBPs bind early in gut development, before feeding. Later in development, VCBPs and bacteria colocalize to chitin-rich mucus along the intestinal wall. VCBP-C influences biofilm formation in vitro and, collectively, the findings of this study suggest that VCBP-C may influence the overall settlement and colonization of bacteria in the Ciona gut. Basic relationships between soluble immunoglobulin-type molecules, endogenous chitin and bacteria arose early in chordate evolution and are integral to the overall function of the gut barrier.

Allorecognition in Hydractinia, a cnidarian, is governed by two different, highly polymorphic genes encoding transmembrane proteins. Using a fluorescent cell read-out system, a new study now shows that the basis for specificity involves homophilic interactions between extracellular domains., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The molecules that mediate innate immunity are encoded by relatively few genes and exhibit broad specificity. Detailed annotation of the Pacific oyster (Crassostrea gigas) genome, a protostome invertebrate, reveals large-scale duplication and divergence of multigene families encoding molecules that effect innate immunity. Transcriptome analyses indicate dynamic and orchestrated specific expression of numerous innate immune genes in response to experimental challenge with pathogens, including bacteria, and a pathogenic virus. Variable expression of individual members of the multigene families encoding these genes also occurs during different types of abiotic stress (environmentally-equivalent conditions of temperature, salinity and desiccation). Multiple families of immune genes are responsive in concert to certain biotic and abiotic challenges. Individual members of expanded families of immune genes are differentially expressed under both biotic challenge and abiotic stress conditions. Members of the same families of innate immune molecules also are transcribed in developmental stage- and tissue-specific manners. An integrated, highly complex innate immune system that exhibits remarkable discriminatory properties and responses to different pathogens as well as environmental stress has arisen through the adaptive recruitment of tandem duplicated genes. The co-adaptive evolution of stress and innate immune responses appears to have an ancient origin in phylogeny.

A variety of germline and somatic immune mechanisms have evolved in vertebrate and invertebrate species to detect a wide array of pathogenic invaders. The gut is a particularly significant site in terms of distinguishing pathogens from potentially beneficial microbes. Ciona intestinalis, a filter-feeding marine protochordate that is ancestral to the vertebrate form, possesses variable region-containing chitin-binding proteins (VCBPs), a family of innate immune receptors, which recognize bacteria through an immunoglobulin-type variable region. The manner in which VCBPs mediate immune recognition appears to be related to the development and bacterial colonization of the gut, and it is likely that these molecules are critical elements in achieving overall immune and physiological homeostasis.

The animal gut serves as a primary location for the complex host-microbe interplay that is essential for homeostasis and may also reflect the types of ancient selective pressures that spawned the emergence of immunity in metazoans. In this review, we present a phylogenetic survey of gut host-microbe interactions and suggest that host defense systems arose not only to protect tissue directly from pathogenic attack but also to actively support growth of specific communities of mutualists. This functional dichotomy resulted in the evolution of immune systems much more tuned for harmonious existence with microbes than previously thought, existing as dynamic but primarily cooperative entities in the present day. We further present the protochordate Ciona intestinalis as a promising model for studying gut host-bacterial dialogue. The taxonomic position, gut physiology and experimental tractability of Ciona offer unique advantages in dissecting host-microbe interplay and can complement studies in other model systems., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We have analyzed the available genome and transcriptome resources from the coelacanth in order to characterize genes involved in adaptive immunity. Two highly distinctive IgW-encoding loci have been identified that exhibit a unique genomic organization, including a multiplicity of tandemly repeated constant region exons. The overall organization of the IgW loci precludes typical heavy chain class switching. A locus encoding IgM could not be identified either computationally or by using several different experimental strategies. Four distinct sets of genes encoding Ig light chains were identified. This includes a variant sigma-type Ig light chain previously identified only in cartilaginous fishes and which is now provisionally denoted sigma-2. Genes encoding α/β and γ/δ T-cell receptors, and CD3, CD4, and CD8 co-receptors also were characterized. Ig heavy chain variable region genes and TCR components are interspersed within the TCR α/δ locus; this organization previously was reported only in tetrapods and raises questions regarding evolution and functional cooption of genes encoding variable regions. The composition, organization and syntenic conservation of the major histocompatibility complex locus have been characterized. We also identified large numbers of genes encoding cytokines and their receptors, and other genes associated with adaptive immunity. In terms of sequence identity and organization, the adaptive immune genes of the coelacanth more closely resemble orthologous genes in tetrapods than those in teleost fishes, consistent with current phylogenomic interpretations. Overall, the work reported described herein highlights the complexity inherent in the coelacanth genome and provides a rich catalog of immune genes for future investigations., (© 2014 Wiley Periodicals, Inc.)

Five large multigene families encoding innate-type immune receptors that are comprised of immunoglobulin domains have been identified in bony fish, of which four do not possess definable mammalian orthologs. The members of some of the multigene families exhibit unusually extensive patterns of divergence and the individual family members demonstrate marked variation in interspecific comparisons. As a group, the gene families reveal striking differences in domain type and content, mechanisms of intracellular signaling, basic structural features, haplotype and allelic variation and ligand binding. The potential functional roles of these innate immune receptors, their relationships to immune genes in higher vertebrate species and the basis for their adaptive evolution are of broad interest. Ongoing investigations are expected to provide new insight into alternative mechanisms of immunity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Variable region-containing chitin-binding proteins (VCBPs) are secreted, immune-type molecules that have been described in both amphioxus, a cephalochordate, and sea squirt, Ciona intestinalis, a urochordate. In adult Ciona, VCBP-A, -B and -C are expressed in hemocytes and the cells of the gastrointestinal tract. VCBP-C binds bacteria in the stomach lumen and functions as an opsonin in vitro. In the present paper the expression of VCBPs has been characterized during development using in situ hybridization, immunohistochemical staining and quantitative polymerase chain reaction (qPCR) technologies. The expression of VCBP-A and -C is detected first in discrete areas of larva endoderm and becomes progressively localized during differentiation in the stomach and intestine, marking the development of gut tracts. In "small adults" (1-2 cm juveniles) expression of VCBP-C persists and VCBP-A gradually diminishes, ultimately replaced by expression of VCBP-B. The expression of VCBP-A and -C in stage 7-8 juveniles, at which point animals have already started feeding, is influenced significantly by challenge with either Gram-positive or -negative bacteria. A potential role for VCBPs in gut-microbiota interactions and homeostasis is indicated.

It is now widely understood that all animals engage in complex interactions with bacteria (or microbes) throughout their various life stages. This ancient exchange can involve cooperation and has resulted in a wide range of evolved host-microbial interdependencies, including those observed in the gut. Ciona intestinalis, a filter-feeding basal chordate and classic developmental model that can be experimentally manipulated, is being employed to help define these relationships. Ciona larvae are first exposed internally to microbes upon the initiation of feeding in metamorphosed individuals; however, whether or not these microbes subsequently colonize the gut and whether or not Ciona forms relationships with specific bacteria in the gut remains unknown. In this report, we show that the Ciona gut not only is colonized by a complex community of bacteria, but also that samples from three geographically isolated populations reveal striking similarity in abundant operational taxonomic units (OTUs) consistent with the selection of a core community by the gut ecosystem.

The polymeric immunoglobulin (Ig) receptor (pIgR) is an integral transmembrane glycoprotein that plays an important role in the mammalian immune response by transporting soluble polymeric Igs across mucosal epithelial cells. Single pIgR genes, which are expressed in lymphoid organs including mucosal tissues, have been identified in several teleost species. A single pigr gene has been identified on zebrafish chromosome 2 along with a large multigene family consisting of 29 pigr-like (PIGRL) genes. Full-length transcripts from ten different PIGRL genes that encode secreted and putative inhibitory membrane-bound receptors have been characterized. Although PIGRL and pigr transcripts are detected in immune tissues, only PIGRL transcripts can be detected in lymphoid and myeloid cells. In contrast to pIgR which binds Igs, certain PIGRL proteins bind phospholipids. PIGRL transcript levels are increased after infection with Streptococcus iniae, suggesting a role for PIGRL genes during bacterial challenge. Transcript levels of PIGRL genes are decreased after infection with Snakehead rhabdovirus, suggesting that viral infection may suppress PIGRL function.

In the colonial tunicate Botryllus schlosseri, a co-dominant trait determines the capacity of adjacent colonies to fuse or reject. An innovative RNA sequencing approach has now identified the gene that predicts the outcomes of this naturally occurring allograft., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.

Complex symbiotic interactions at the surface of host epithelia govern most encounters between host and microbe. The epithelium of the gut is a physiologically ancient structure that is comprised of a single layer of cells and is thought to possess fully developed immunological capabilities. Ciona intestinalis (sea squirt), which is a descendant of the last common ancestor of all vertebrates, is a potentially valuable model for studying barrier defenses and gut microbial immune interactions. A variety of innate immunological phenomena have been well characterized in Ciona, of which many are active in the gut tissues. Interactions with gut microbiota likely involve surface epithelium, secreted immune molecules including variable region-containing chitin-binding proteins, and hemocytes from a densely populated laminar tissue space. The microbial composition of representative gut luminal contents has been characterized by molecular screening and a potentially relevant, reproducible, dysbiosis can be induced via starvation. The dialog between host and microbe in the gut can be investigated in Ciona against the background of a competent innate immune system and in the absence of the integral elements and processes that are characteristic of vertebrate adaptive immunity.

A heretofore-unrecognized multigene family encoding diverse immunoglobulin (Ig) domain-containing proteins (DICPs) was identified in the zebrafish genome. Twenty-nine distinct loci mapping to three chromosomal regions encode receptor-type structures possessing two classes of Ig ectodomains (D1 and D2). The sequence and number of Ig domains, transmembrane regions and signaling motifs vary between DICPs. Interindividual polymorphism and alternative RNA processing contribute to DICP diversity. Molecular models indicate that most D1 domains are of the variable (V) type; D2 domains are Ig-like. Sequence differences between D1 domains are concentrated in hypervariable regions on the front sheet strands of the Ig fold. Recombinant DICP Ig domains bind lipids, a property shared by mammalian CD300 and TREM family members. These findings suggest that novel multigene families encoding diversified immune receptors have arisen in different vertebrate lineages and affect parallel patterns of ligand recognition that potentially impact species-specific advantages., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Immune systems evolve as essential strategies to maintain homeostasis with the environment, prevent microbial assault and recycle damaged host tissues. The immune system is composed of two components, innate and adaptive immunity. The former is common to all animals while the latter consists of a vertebrate-specific system that relies on somatically derived lymphocytes and is associated with near limitless genetic diversity as well as long-term memory. Deuterostome invertebrates provide a view of immune repertoires in phyla that immediately predate the origins of vertebrates. Genomic studies in amphioxus, a cephalochordate, have revealed homologs of genes encoding most innate immune receptors found in vertebrates; however, many of the gene families have undergone dramatic expansions, greatly increasing the innate immune repertoire. In addition, domain-swapping accounts for the innovation of new predicted pathways of receptor function. In both amphioxus and Ciona, a urochordate, the VCBPs (variable region containing chitin-binding proteins), which consist of immunoglobulin V (variable) and chitin binding domains, mediate recognition through the V domains. The V domains of VCBPs in amphioxus exhibit high levels of allelic complexity that presumably relate to functional specificity. Various features of the amphioxus immune repertoire reflect novel selective pressures, which likely have resulted in innovative strategies. Functional genomic studies underscore the value of amphioxus as a model for studying innate immunity and may help reveal how unique relationships between innate immune receptors and both pathogens and symbionts factored in the evolution of adaptive immune systems.

The novel immune-type receptors (NITRs), which have been described in numerous bony fish species, are encoded by multigene families of inhibitory and activating receptors and are predicted to be functional orthologs to the mammalian natural killer cell receptors (NKRs). Within the zebrafish NITR family, nitr9 is the only gene predicted to encode an activating receptor. However, alternative RNA splicing generates three distinct nitr9 transcripts, each of which encodes a different isoform. Although nitr9 transcripts have been detected in zebrafish lymphocytes, the specific hematopoietic lineage(s) that expresses Nitr9 remains to be determined. In an effort to better understand the role of NITRs in zebrafish immunity, anti-Nitr9 monoclonal antibodies were generated and evaluated for the ability to recognize the three Nitr9 isoforms. The application of these antibodies to flow cytometry should prove to be useful for identifying the specific lymphocyte lineages that express Nitr9 and may permit the isolation of Nitr9-expressing cells that can be directly assessed for cytotoxic (e.g., NK) function.

CD300, triggering receptor expressed on myeloid cells (TREM), and TREM-like (TREML) receptors are important regulators of the mammalian immune response. Homologs of these receptors, which occur in activating and inhibitory transmembrane forms as well as soluble variants, are found throughout the jawed vertebrates. Specific ligands for most members of these receptor families remain elusive. We report here that at least 11 separate receptors from the CD300, TREM, and TREML families engage in robust and specific interactions with major polar lipids found in prokaryotic and eukaryotic cell membranes. Both soluble and membrane-bound receptor forms exhibit lipid interactions in the solid phase as well as in a physiological signaling context. Overlapping but distinctive patterns of receptor specificity suggest that the CD300/TREM system as a whole may discriminate immunological stimuli based on lipid signatures, thereby influencing downstream responses.

A number of different classes of molecules function as structural matrices for effecting innate and adaptive immunity. The most extensively characterized mediators of adaptive immunity are the immunoglobulins and T-cell antigen receptors found in jawed vertebrates. In both classes of molecules, unique receptor specificity is effected through somatic variation in the variable (V) structural domain. V region-containing chitin-binding proteins (VCBPs) consist of two tandem Ig V domains as well as a chitin-binding domain. VCBPs are encoded at four loci (i.e., VCBPA-VCBPD) in Ciona, a urochordate, and are expressed by distinct epithelial cells of the stomach and intestine, as well as by granular amoebocytes present in the lamina propria of the gut and in circulating blood. VCBPs are secreted into the gut lumen, and direct binding to bacterial surfaces can be detected by immunogold analysis. Affinity-purified native and recombinant VCBP-C, as well as a construct consisting only of the tandem V domains, enhance bacterial phagocytosis by granular amoebocytes in vitro. Various aspects of VCBP expression and function suggest an early origin for the key elements that are central to the dialogue between the immune system of the host and gut microflora.

Natural killer (NK) cells affect a form of innate immunity that recognizes and eliminates cells that are infected with certain viruses or have undergone malignant transformation. In mammals, this recognition can be mediated through immunoglobulin- (Ig) and/or lectin-type NK receptors (NKRs). NKR genes in mammals range from minimally polymorphic single-copy genes to complex multigene families that exhibit high levels of haplotypic complexity and exhibit significant interspecific variation. Certain single-copy NKR genes that are present in one mammal are present as expanded multigene families in other mammals. These observations highlight NKRs as one of the most rapidly evolving eukaryotic gene families and likely reflect the influence of pathogens, especially viruses, on their evolution. Although well characterized in human and mice, cytotoxic cells that are functionally similar to NK cells have been identified in species ranging from birds to reptiles, amphibians and fish. Although numerous receptors have been identified in non-mammalian vertebrates that share structural relationships with mammalian NKRs, functionally defining these lower vertebrate molecules as NKRs is confounded by methodological and interpretive complexities. Nevertheless, several lines of evidence suggest that NK-type function or its equivalent has sustained a long evolutionary history throughout vertebrate species.

Understanding the transcriptome, defined as the complete transcriptional component of the genome, is far more complex than originally considered. Even with the near fully resolved human and mouse genomes, for which extensive databases of transcribed sequence data (e.g., expressed sequence tags) are available, it is presently not possible to experimentally recover or computationally predict the full range of transcription products that derive from multiexon genes. Many genes are tightly regulated, which could include alternative processing of RNA, and lead to significant underrepresentation of many transcripts. A multitude of factors in addition to cell lineage- and developmental stage-specific expression as well as shortcomings in computational methods result in a less than complete understanding of transcriptional complexity. Here, we describe an approach to predict and evaluate a more complete repertoire of transcriptional products that derive from specific genetic loci with attention toward analysis of immune receptor genes. This approach is particularly useful in identifying gene products, including alternative splice forms, that originate from complex multigene families.

Recombinant fusion proteins incorporating experimental protein domains fused to immunoglobulin Fc regions have become widely utilized in studies of protein-ligand interactions. The advantages of these systems include an inherent increase in avidity provided by the multimerization of Fc regions, combined with robust detection methods based on numerous commercially available secondary reagents directed against the Fc tag. We describe a set of methods for subcloning, expression, and purification of chimeric protein reagents containing a protein domain (or domains) of interest fused to a C-terminal moiety derived from the Fc region of either IgG or IgM.

Innate immune gene repertoires are restricted primarily to germline variation. Adaptive immunity, by comparison, relies on somatic variation of germline-encoded genes to generate extraordinarily large numbers of non-heritable antigen recognition motifs. Invertebrates lack the key features of vertebrate adaptive immunity, but have evolved a variety of alternative mechanisms to successfully protect the integrity of "self"; in many cases, these appear to be taxon-specific innovations. In the protochordate Branchiostoma floridae (amphioxus), the variable region-containing chitin-binding proteins (VCBPs) constitute a multigene family (comprised of VCBPs 1-5), which possesses features that are consistent with innate immune-type function. A large number of VCBP alleles and haplotypes are shown to exhibit levels of polymorphism exceeding the elevated overall levels determined for the whole amphioxus genome (JGI). VCBP genes of the 2 and 5 types are distinguished further by a highly polymorphic segment (exon 2) in the N-terminal immunoglobulin domain, defined previously as a "hypervariable region" or a "hotspot." Genomic deoxyribonucleic acid (DNA) and complementary DNA (cDNA) sequences from large numbers of animals representing different populations reveal further significant differences in sequence complexity within and across VCBP2/5 haplotypes that arise through overlapping mechanisms of genetic exchange, gene copy number variation as well as mutation and give rise to distinct allelic lineages. The collective observations suggest that mechanisms were in place at the time of divergence of the cephalochordates that could selectively hyperdiversify immune-type receptors within a multigene family.

Adaptive immunity is mediated through numerous genetic and cellular processes that generate favourable somatic variants of antigen-binding receptors under evolutionary selection pressure by pathogens and other factors. Advances in our understanding of immunity in mammals and other model organisms are revealing the underlying basis and complexity of this remarkable system. Although the evolution of adaptive immunity has been thought to occur by the acquisition of novel molecular capabilities, an increasing amount of information from new model systems suggest that co-option and redirection of pre-existing systems are the main source of innovation. We combine evidence from a wide range of organisms to obtain an integrated view of the origins and patterns of divergence in adaptive immunity.

Characterization of immune receptors found in phylogenetically disparate species at the genetic, structural and functional levels has provided unique insight into the evolutionary acquisition of immune function. The roles of variable- and intermediate-type immunoglobulin (Ig) domains in direct recognition of ligands and other functions are far wider than previously anticipated. Common mechanisms of multigene family diversification and expansion as well as unique adaptations that relate to function continue to provide unique insight into the numerous patterns, processes and complex interactions that regulate the host response to infectious challenge., ((c) 2009 Elsevier Ltd. All rights reserved.)

Novel immune-type receptors (NITRs) are encoded by large multi-gene families and share structural and signaling similarities to mammalian natural killer receptors (NKRs). NITRs have been identified in multiple bony fish species, including zebrafish, and may be restricted to this large taxonomic group. Thirty-nine NITR genes that can be classified into 14 families are encoded on zebrafish chromosomes 7 and 14. Herein, we demonstrate the expression of multiple NITR genes in the zebrafish ovary and during embryogenesis. All 14 families of zebrafish NITRs are expressed in hematopoietic kidney, spleen and intestine as are immunoglobulin and T cell antigen receptors. Furthermore, all 14 families of NITRs are shown to be expressed in the lymphocyte lineage, but not in the myeloid lineage, consistent with the hypothesis that NITRs function as NKRs. Sequence analyses of NITR amplicons identify known alleles and reveal additional alleles within the nitr1, nitr2, nitr3, and nitr5 families, reflecting the recent evolution of this gene family.

Alloimmune specificity and histocompatibility, driven by genetic polymorphism, are ancient determinants of self-/non-self-recognition. Recent molecular genetic evidence has revealed an allodeterminant in the cnidarian Hydractinia that consistently predicts histocompatibility reactions.

A recessive nonsense mutation in the zebrafish recombination activating gene 1 (rag1) gene results in defective V(D)J recombination; however, animals homozygous for this mutation (rag1(-/-)) are reportedly viable and fertile in standard, nonsterile aquarium conditions but display increased mortality after intraperitoneal injection with mycobacteria. Based on their survival in nonsterile environments, we hypothesized that the rag1(-/-) zebrafish may possess an "enhanced" innate immune response to compensate for the lack of an adaptive immune system. To test this hypothesis, microarray analyses were used to compare the expression profiles of the intestines and hematopoietic kidneys of rag1 deficient zebrafish to the expression profiles of control (heterozygous) siblings. The expression levels of 12 genes were significantly altered in the rag1(-/-) kidney including the up regulation of a putative interferon stimulated gene, and the down regulation of genes encoding fatty acid binding protein 10, keratin 5 and multiple heat shock proteins. The expression levels of 87 genes were shown to be significantly altered in the rag1(-/-) intestine; the majority of these differences reflect increased expression of innate immune genes, including those of the coagulation and complement pathways. Subsequent analyses of orthologous coagulation and complement genes in Rag1(-/-) mice indicate increased transcription of the complement C4 gene in the Rag1(-/-) intestine.

Background: The variable region-containing chitin-binding proteins (VCBPs) are found in protochordates and consist of two tandem immunoglobulin variable (V)-type domains and a chitin-binding domain. We previously have shown that these polymorphic genes, which primarily are expressed in the gut, exhibit characteristics of immune genes. In this report, we describe VCBP genomic organization and characterize adjacent and intervening genetic features which may influence both their polymorphism and complex transcriptional repertoire., Results: VCBP genes 1, 2, 4, and 5 are encoded in a single contiguous gene-rich chromosomal region and VCBP3 is encoded in a separate locus. The VCBPs exhibit extensive haplotype variation, including copy number variation (CNV), indel polymorphism and a markedly elevated variation in repeat type and density. In at least one haplotype, inverted repeats occur more frequently than elsewhere in the genome. Multi-animal cDNA screening, as well as transcriptional profilingusing a novel transfection system, suggests that haplotype-specific transcriptional variants may contribute to VCBP genetic diversity., Conclusion: The availability of the Branchiostoma floridae genome (Joint Genome Institute, Brafl1), along with BAC and PAC screening and sequencing described here, reveal that the relatively limited number of VCBP genes present in the amphioxus genome exhibit exceptionally high haplotype variation. These VCBP haplotypes contribute a diverse pool of allelic variants, which includes gene copy number variation, pseudogenes, and other polymorphisms, while contributing secondary effects on gene transcription as well.

Novel immune-type receptors (NITRs) comprise an exceptionally large, diversified family of activating and inhibitory receptors that has been identified in bony fish. Here, we characterized the structure of an activating NITR that is expressed by a cytotoxic natural killer (NK)-like cell line and that specifically binds an allogeneic B cell target. A single amino acid residue within the NITR immunoglobulin variable (V)-type domain accounts for specificity of the interaction. Structures solved by X-ray crystallography revealed that the V-type domains of NITRs form homodimers resembling rearranging antigen-binding receptor heterodimers. CDR1 elements of both subunits of NITR dimers form ligand-binding surfaces that determine specificity for the nonself target. In the evolution of immune function, it appears that a specific NK type of innate recognition may be mediated by a complex germline multigene family of V structures resembling those that are somatically diversified in adaptive immunological responses.

Cephalochordates, urochordates, and vertebrates evolved from a common ancestor over 520 million years ago. To improve our understanding of chordate evolution and the origin of vertebrates, we intensively searched for particular genes, gene families, and conserved noncoding elements in the sequenced genome of the cephalochordate Branchiostoma floridae, commonly called amphioxus or lancelets. Special attention was given to homeobox genes, opsin genes, genes involved in neural crest development, nuclear receptor genes, genes encoding components of the endocrine and immune systems, and conserved cis-regulatory enhancers. The amphioxus genome contains a basic set of chordate genes involved in development and cell signaling, including a fifteenth Hox gene. This set includes many genes that were co-opted in vertebrates for new roles in neural crest development and adaptive immunity. However, where amphioxus has a single gene, vertebrates often have two, three, or four paralogs derived from two whole-genome duplication events. In addition, several transcriptional enhancers are conserved between amphioxus and vertebrates--a very wide phylogenetic distance. In contrast, urochordate genomes have lost many genes, including a diversity of homeobox families and genes involved in steroid hormone function. The amphioxus genome also exhibits derived features, including duplications of opsins and genes proposed to function in innate immunity and endocrine systems. Our results indicate that the amphioxus genome is elemental to an understanding of the biology and evolution of nonchordate deuterostomes, invertebrate chordates, and vertebrates.

Background: Novel immune-type receptor (NITR) genes are members of diversified multigene families that are found in bony fish and encode type I transmembrane proteins containing one or two extracellular immunoglobulin (Ig) domains. The majority of NITRs can be classified as inhibitory receptors that possess cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs). A much smaller number of NITRs can be classified as activating receptors by the lack of cytoplasmic ITIMs and presence of a positively charged residue within their transmembrane domain, which permits partnering with an activating adaptor protein., Results: Forty-four NITR genes in medaka (Oryzias latipes) are located in three gene clusters on chromosomes 10, 18 and 21 and can be organized into 24 families including inhibitory and activating forms. The particularly large dataset acquired in medaka makes direct comparison possible to another complete dataset acquired in zebrafish in which NITRs are localized in two clusters on different chromosomes. The two largest medaka NITR gene clusters share conserved synteny with the two zebrafish NITR gene clusters. Shared synteny between NITRs and CD8A/CD8B is limited but consistent with a potential common ancestry., Conclusion: Comprehensive phylogenetic analyses between the complete datasets of NITRs from medaka and zebrafish indicate multiple species-specific expansions of different families of NITRs. The patterns of sequence variation among gene family members are consistent with recent birth-and-death events. Similar effects have been observed with mammalian immunoglobulin (Ig), T cell antigen receptor (TCR) and killer cell immunoglobulin-like receptor (KIR) genes. NITRs likely diverged along an independent pathway from that of the somatically rearranging antigen binding receptors but have undergone parallel evolution of V family diversity.

Novel immune-type receptors (NITRs) are immunoglobulin-variable (V) domain-containing cell surface proteins that possess characteristic activating/inhibitory signaling motifs and are expressed in hematopoietic cells. NITRs are encoded by multigene families and have been identified in bony fish species. A single gene cluster, which encodes 36 NITRs that can be classified into 12 families, has been mapped to zebrafish chromosome 7. We report herein the presence of a second NITR gene cluster on zebrafish chromosome 14, which is comprised of three genes (nitr13, nitr14a, and nitr14b) representing two additional NITR gene families. Phylogenetic analyses indicate that the V domains encoded by the nitr13 and nitr14 genes are more similar to each other than any other zebrafish NITR suggesting that these genes arose from a tandem gene duplication event. Similar analyses comparing zebrafish Nitr13 and Nitr14 to NITRs from other fish species indicate that the nitr13 and nitr14 genes are phylogenetically related to the catfish IpNITR13 and IpNITR15 genes. Sequence features of the chromosomal region encoding nitr13 suggest that this gene arose via retrotransposition.

Background: Regulation in protein networks often utilizes specialized domains that 'join' (or 'connect') the network through specific protein-protein interactions. The innate immune system, which provides a first and, in many species, the only line of defense against microbial and viral pathogens, is regulated in this way. Amphioxus (Branchiostoma floridae), whose genome was recently sequenced, occupies a unique position in the evolution of innate immunity, having diverged within the chordate lineage prior to the emergence of the adaptive immune system in vertebrates., Results: The repertoire of several families of innate immunity proteins is expanded in amphioxus compared to both vertebrates and protostome invertebrates. Part of this expansion consists of genes encoding proteins with unusual domain architectures, which often contain both upstream receptor and downstream activator domains, suggesting a potential role for direct connections (shortcuts) that bypass usual signal transduction pathways., Conclusion: Domain rearrangements can potentially alter the topology of protein-protein interaction (and regulatory) networks. The extent of such arrangements in the innate immune network of amphioxus suggests that domain shuffling, which is an important mechanism in the evolution of multidomain proteins, has also shaped the development of immune systems.

X-ray diffraction data from crystals of a novel immune-type receptor (NITR10 from the catfish Ictalurus punctatus) were collected to 1.65 A resolution and reduced to the primitive hexagonal lattice. Native and selenomethionine derivatives of NITR10 crystallized under different conditions yielded P3(1)21 crystals. SeMet NITR10 was phased to a correlation coefficient of 0.77 by SAD methods and experimental electron-density maps were calculated to 1.65 A. Five NITR10 molecules are predicted to be present in the asymmetric unit based on the Matthews coefficient.

Transmembrane bound receptors comprised of extracellular immunoglobulin (Ig) or lectin domains play integral roles in a large number of immune functions including inhibitory and activating responses. The function of many of the activating receptors requires a physical interaction with an adaptor protein possessing a cytoplasmic regulatory motif. The partnering of an activating receptor with an adaptor protein relies on complementary charged residues in the two transmembrane domains. The mammalian natural killer (NK) and Fc receptors (FcR) represent two of many receptor families, which possess activating receptors that partner with adaptor proteins for signaling. Zebrafish represent a powerful experimental model for understanding developmental regulation at early stages of embryogenesis and for efficiently generating transgenic animals. In an effort to understand developmental aspects of immune receptor function, we have accessed the partially annotated zebrafish genome to identify six different adaptor molecules: Dap10, Dap12, Cd3zeta, Cd3zeta-like, FcRgamma and FcRgamma-like that are homologous to those effecting immune function in mammals. Their genomic organizations have been characterized, cDNA transcripts have been recovered, phylogenetic relationships have been defined and their cell lineage-specific expression patterns have been established.

Our views of both innate and adaptive immunity have been significantly modified by recent studies of immune receptors and immunity in protostomes, invertebrate deuterostomes, and jawless vertebrates. Extraordinary variation in the means whereby organisms recognize pathogens has been revealed by a series of recent findings, including: novel forms of familiar immune receptors, high genetic polymorphism for new receptor types, germline rearrangement for non-Ig domain receptors, somatic variation of germline-encoded receptors, and unusually complex alternative splicing of genes with both immune and non-immune roles. Collectively, these observations underscore heretofore unrecognized pathways in the evolution of immune recognition and suggest universal processes by which immune systems co-opt and integrate existing cellular mechanisms to effect diverse recognition functions.

Both inhibitory and activating forms of natural killer (NK) cell receptors are found in mammals. The activating receptors play a direct role in the recognition of virally infected or transformed cells and transduce activating signals into the cell by partnering with an adaptor protein, which contains a cytoplasmic activation motif. Activating NK receptors encoded by the mammalian leukocyte receptor complex (e.g., killer cell immunoglobulin-like receptors) and the natural killer complex (e.g., Ly49s) partner with the adaptor protein DAP12, whereas NK receptors encoded in the CD94/NKG2 complex partner with the adaptor protein DAP10. Novel immune-type receptors (NITRs) found in bony fish share several common features with immunoglobulin-type NK receptors. Nitr9 is a putative activating receptor in zebrafish that induces cytotoxicity within the context of human NK cells. One isoform of Nitr9, Nitr9L, is shown here to preferentially partner with a zebrafish ortholog of Dap12. Cross-linking the Nitr9L-Dap12 complex results in activation of the phosphytidylinositol 3-kinase-->AKT-->extracellular signal-regulated kinase pathway suggesting that the DAP12-based activating pathway is conserved between bony fish and mammals.