To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before the teleost genome duplication (TGD). The slowly evolving gar genome 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 (e.g., Hox, ParaHox, and miRNA genes). Numerous conserved non-coding elements (CNEs, often cis-regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles of such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses revealed that the sum of expression domains and levels from duplicated teleost genes often approximate patterns and levels of 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.

A series of products were amplified using a PCR strategy based on short minimally degenerate primers and R. eglanteria (clearnose skate) spleen cDNA as template. These products were used as probes to select corresponding cDNAs from a spleen cDNA library. The cDNA sequences exhibit significant identity with prototypic (alpha, beta, gamma, and delta T cell antigen receptor (TCR) genes. Characterization of cDNAs reveals extensive variable region diversity, putative diversity segments, and varying degrees of junctional diversification. This demonstrates expression of both alpha/beta and gamma/delta TCR genes at an early level of vertebrate phylogeny and indicates that the three major known classes of rearranging antigen receptors were present in the common ancestor of the present-day jawed vertebrates.

The immunoglobulin (Ig) heavy chain variable (VH) gene family of Heterodontus francisci (horned shark), a phylogenetically distant vertebrate, is unique in that VH, diversity (DH), joining (JH) and constant region (CH) gene segments are linked closely, in multiple individual clusters. The V regions of 12 genomic (liver and gonad) DNA clones have been sequenced completely and three organization patterns are evident: (i) VH‐D1‐D2‐JH‐CH with unique 12/22 and 12/12 spacers in the respective D recombination signal sequences (RSSs); VH and JH segments have 23 nucleotide (nt) spacers, (ii) VHDH‐JH‐CH, an unusual germline configuration with joined VH and DH segments and (iii) VHDHJH‐CH, with all segmental elements being joined. The latter two configurations do not appear to be pseudogenes. Another VH‐D1‐D2‐JH‐CH gene possesses a D1 segment that is flanked by RSSs with 12 nt spacers and a D2 segment with 22/12 spacers. Based on the comparison of spleen, VH+ cDNA sequences to a germline consensus, it is evident that both DH segments as well as junctional and N‐type diversity account for Ig variability. In this early vertebrate, the Ig genes share unique properties with higher vertebrate T‐cell receptor as well as with Ig and may reflect the structure of a common ancestral antigen binding receptor gene.

The novel immune-type receptor (NITR) genes encode a unique multigene family of leukocyte regulatory receptors, which possess an extracellular Ig variable (V) domain and may function in innate immunity. Artificial chromosomes that encode zebrafish NITRs have been assembled into a contig spanning Ϸ350 kb. Resolution of the complete NITR gene cluster has led to the identification of eight previously undescribed families of NITRs and has revealed the presence of C-type lectins within the locus. A maximum haplotype of 36 NITR genes (138 gene sequences in total) can be grouped into 12 distinct families, including inhibitory and activating receptors. An extreme level of interindividual heterozygosity is reflected in allelic polymorphisms, haplotype variation, and familyspecific isoform complexity. In addition, the exceptional diversity of NITR sequences among species suggests divergent evolution of this multigene family with a birth-and-death process of member genes. High-confidence modeling of Nitr V-domain structures reveals a significant shift in the spatial orientation of the Ig fold, in the region of highest interfamily variation, compared with Ig V domains. These studies resolve a complete immune gene cluster in zebrafish and indicate that the NITRs represent the most complex family of activating͞inhibitory surface receptors thus far described.

Antigen recognition in the adaptive immune response by Ig and T-cell antigen receptors (TCRs) is effected through patterned differences in the peptide sequence in the V regions. V-region specificity forms through genetically programmed rearrangement of individual, diversified segmental elements in single somatic cells. Other Ig superfamily members, including natural killer receptors that mediate cell-surface recognition, do not undergo segmental reorganization, and contain type-2 C (C2) domains, which are structurally distinct from the C1 domains found in Ig and TCR. Immunoreceptor tyrosine-based inhibitory motifs that transduce negative regulatory signals through the cell membrane are found in certain natural killer and other cell surface inhibitory receptors, but not in Ig and TCR. In this study, we employ a genomic approach by using the pufferfish (Spheroides nephelus) to characterize a nonrearranging novel immune-type receptor gene family. Twentysix different nonrearranging genes, which each encode highly diversified V as well as a V-like C2 extracellular domain, a transmembrane region, and in most instances, an immunoreceptor tyrosine-based inhibitory motif-containing cytoplasmic tail, are identified in an Ϸ113 kb P1 artificial chromosome insert. The presence in novel immune-type receptor genes of V regions that are related closely to those found in Ig and TCR as well as regulatory motifs that are characteristic of inhibitory receptors implies a heretofore unrecognized link between known receptors that mediate adaptive and innate immune functions.V gene diversity ͉ immunoreceptor tyrosine-based inhibitory motif ͉ evolution ͉ adaptive immunity ͉ innate immunity I g and T-cell antigen receptor (TCR) genes are the primary mediators of highly specific adaptive immune responses. Recognition of antigens by these two structurally related but functionally distinct types of antigen-binding receptors is achieved through specific polypeptide folding patterns in N-terminal V regions that are created by both somatic rearrangement of individual segmental elements encoding V, diversity (D), and J regions as well as through nontemplated mechanisms that introduce additional sequence variation (1, 2). Patterns of shared sequence identity, organizational similarities, and a common rearrangement mechanism in Ig and TCR found in jawed vertebrate species are consistent with their origin from a common ancestral form in the distant evolutionary past and diversification in structure and organization throughout vertebrate phylogeny (3). Although V regions have undergone diversification during vertebrate evolution, comparisons of both Ig and TCR, as well as CD8␤, a nonrearranging V region-containing gene expressed on the surface of T cells (4, 5), indicate general conservation of short-sequence motifs in the second and third framework regions (FR2 and FR3) of the V region (6). Sharing of such short-sequence regions forms the basis for a strategy that has been used to identify TCR gene homologs as well as Ig genes and an Ig-like gene ...

E ver-increasing interest is being directed to the role of innate processes in the immune system (1). Particular attention has been given recently to the group of genes that includes the natural killer cell (NK) receptors that are encoded at the leukocyte receptor cluster (LRC) locus on human chromosome 19q13.4 and similar receptors on mouse chromosome 7. These genes, which include killer Ig-like receptors (KIR), paired Ig-like receptors, Ig-like transcripts (ILT) [leukocyte inhibitory receptors (LIR) and monocyte inhibitory receptors (MIR)], leukocyte-associated inhibitory receptor genes, and NKp46͞MAR-1 (2-8), constitute a subset of the Ig gene superfamily (IgSF), and at least some members mediate innate recognition (9). The present understanding of LRC genes is based on studies in mammals; however, it is presumed that they arose early in vertebrate phylogeny.On the basis of considerations of both structural specialization and genetic complexity, it is possible that the Ig and T cell antigen receptor (TCR) multigene families, which undergo segmental reorganization and mediate adaptive immunity, arose from innate immune precursor receptors that are not associated with somatic reorganization. The ability of the Ig and TCR subset of the IgSF to recognize a multitude of antigens relies on extensive diversity in their variable (V) regions and is achieved through site-specific rearrangement of V, diversity (D), and joining (J) segmental elements as well as through additional somatic variation and ultimately somatic hypermutation. Within the V region, diversity in both families of antigen receptors is concentrated in complementarity determining regions (CDRs); variation in CDR1 and CDR2 is encoded in the germ line, whereas variation in CDR3 originates somatically. No evidence has been found for structural features or somatic reorganization associated with V regions in known members of the LRC.A third family of diversified V region-containing receptors, termed novel immune-type receptor (NITR) genes, was identified recently in the compact genome of the pufferfish (10). V regions of NITR genes in this species are organized in families, exhibit variation in CDR1 and CDR2, and like Ig and TCR genes encode J regions but neither rearrange nor appear to exhibit other forms of somatic variation (11). In an overall sense, NITRs are structurally similar to certain members of the LRC in that they possess two extracellular Ig domains, a transmembrane region and a cytoplasmic tail, which contains immunoreceptor tyrosine-based inhibition motifs (ITIMs) that function in negative signal transduction pathways. NITRs possess features of both adaptive and innate immune receptors, and their consideration is significant in addressing the evolution of immune function (12).Despite the utility of the pufferfish genome in terms of the initial identification of NITR genes, this model system is limited in terms of further defining the genetics, developmental regulation, cell lineage-specific expression, and function of NITRs. To address these ...

Immune inhibitory receptor genes that encode a variable (V) region, a unique V-like C2 (V͞C2) domain, a transmembrane region, and a cytoplasmic tail containing immunoreceptor tyrosine-based inhibition motifs (ITIMs) have been described previously in two lineages of bony fish. In the present study, eleven related genes encoding distinct structural forms have been identified in Ictalurus punctatus (channel catfish), a well characterized immunological model system that represents a third independent bony fish lineage. Each of the different genes encodes an N-terminal V region but differs in the number of extracellular Ig domains, number and location of joining (J) region-like motifs, presence of transmembrane regions, presence of charged residues in transmembrane regions, presence of cytoplasmic tails, and͞or distribution of ITIM(s) within the cytoplasmic tails. Variation in the numbers of genomic copies of the different gene types, their patterns of expression, and relative levels of expression in mixed leukocyte cultures (MLC) is reported. V region-containing immune-type genes constitute a far more complex family than recognized originally and include individual members that might function in inhibitory or, potentially activatory manners. E xtended multigene families belonging to the Ig gene superfamily (IgSF) account for a diverse range of immunological functions including recognition of antigens and antigenic peptides by both somatically rearranging Ig and T cell antigen receptor (TCR) genes, as well as by major histocompatibility complex (MHC) molecules. The origins of the three diverse systems of effector molecules can be traced through analyses of these genes in extant species of representative early, jawed vertebrates (1). Recently, multigene families which encode novel immune-type receptors (NITR͞nitr) have been described in Spheroides nephelus (Southern pufferfish; ref. 2) and Danio rerio (zebrafish; ref. 3). The NITR genes described in these species encode two extracellular Ig domains [a variable (V) domain and a V-like C2 (V͞C2) domain], a transmembrane region, and most often, immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic tail. The general structural characteristics of the NITR V domain are common to the corresponding regions of both Ig and TCR (4); whereas ITIMs are found in several inhibitory receptors, which are encoded at the leukocyte receptor cluster (LRC) on human chromosome 19q13.3-13.4 and at a corresponding location on mouse chromosome 7 and include natural killer (NK) receptors, such as killer cell Ig-type receptors (KIRs) (5). Unlike NITR genes, LRC genes do not encode V regions. A number of questions arise regarding the distribution of the NITR genes in vertebrate phylogeny, their function, and the relatedness of NITR genes to other families of genes that are involved in immune function, specifically, the immune inhibitory receptors of the mammalian LRC.The lack of immunologically relevant in vitro culture systems in pufferfish and zebrafish severely limits fun...

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 varies 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 effect parallel patterns of ligand recognition that potentially impact species-specific advantages.