The spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons.

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.

Genome complexity in the coelacanth is reflected in its adaptive immune system.

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/ß 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.

Atypical RNAs in the coelacanth transcriptome.

Circular and apparently trans-spliced RNAs have recently been reported as abundant types of transcripts in mammalian transcriptome data. Both types of non-colinear RNAs are also abundant in RNA-seq of different tissue from both the African and the Indonesian coelacanth. We observe more than 8,000 lincRNAs with normal gene structure and several thousands of circularized and trans-spliced products, showing that such atypical RNAs form a substantial contribution to the transcriptome. Surprisingly, the majority of the circularizing and trans-connecting splice junctions are unique to atypical forms, that is, are not used in normal isoforms.

Evolution of adaptive immune recognition in jawless vertebrates.

All extant vertebrates possess an adaptive immune system wherein diverse immune receptors are created and deployed in specialized blood cell lineages. Recent advances in DNA sequencing and developmental resources for basal vertebrates have facilitated numerous comparative analyses that have shed new light on the molecular and cellular bases of immune defense and the mechanisms of immune receptor diversification in the "jawless" vertebrates. With data from these key species in hand, it is becoming possible to infer some general aspects of the early evolution of vertebrate adaptive immunity. All jawed vertebrates assemble their antigen-receptor genes through combinatorial recombination of different "diversity" segments into immunoglobulin or T-cell receptor genes. However, the jawless vertebrates employ an analogous, but independently derived set of immune receptors in order to recognize and bind antigens: the variable lymphocyte receptors (VLRs). The means by which this locus generates receptor diversity and achieves antigen specificity is of considerable interest because these mechanisms represent a completely independent strategy for building a large immune repertoire. Therefore, studies of the VLR system are providing insight into the fundamental principles and evolutionary potential of adaptive immune recognition systems. Here we review and synthesize the wealth of data that have been generated towards understanding the evolution of the adaptive immune system in the jawless vertebrates.

Evolution and development of immunological structures in the lamprey.

Comparative immunology has been revitalized by the integration of genomics approaches, which allow a foothold into addressing problems that previously had been difficult to study. One such problem had been the enigmatic finding of overt immune anatomical structures in the lamprey, yet its apparent lack of bona fide immunoglobulin or T cell receptor molecules. The genomic characterization of a novel extended locus that undergoes rearrangements to generate receptor diversity and the subsequent implementation of this diversity in the immune system of lampreys have generated considerable interest as well as new avenues for investigation. Here, we review the anatomical structures of the lamprey that exhibit lympho-hematopoietic characteristics, with the ultimate goal of reconciling these data with contemporary molecular findings. By integrating these datasets we seek to better understand how an alternative adaptive immune system could have evolved.

Description of a fugu CXC chemokine and two CXC receptor genes, and characterization of the effects of different stimulators on their expression.

The primary structures of a CXC chemokine (CXCL8) and two CXC receptors (CXCR) have been characterized in fugu, Takifugu rubripes. Unlike mammalian and avian species, CXCL8 of teleosts including fugu lacks the ELR motif that appears to be important in ligand/receptor interactions on neutrophils. Genomic organization shows that fugu CXCL8 gene consists of four exons and three introns. As in other vertebrates, two CXCR genes isolated from fugu encode proteins CXCR1 and CXCR2 that possess characteristic seven transmembrane domains. Each receptor consists of two exons separated by an intron. Synteny analysis indicates that these two CXCRs were derived from whole genome duplication in teleosts, differing from mammalian CXCR1 and CXCR2. All of these genes are primarily expressed in the lymphoid tissues. Immune stimulation with PHA showed that the expression of both CXCL8 and CXCRs in PBL are upregulated even after only a short time period, but downregulated by LPS stimulation, implying that these genes are involved in the regulation of the immune response in fugu.

A teleost polymeric Ig receptor exhibiting two Ig-like domains transports tetrameric IgM into the skin.

The skin mucus IgM is an important molecule in the mucosal immune system of teleost skin. However, the transport mechanism associated with this molecule has yet to be clarified. In this study, we isolated a gene encoding a polymeric Ig receptor (pIgR) from a species of teleost fish, Takifugu rubripes (fugu). This gene is known to be an Ig transporter in the intestine of mammals. Our studies further demonstrated that fugu pIgR was expressed in the skin and that a fragment of pIgR bound to tetrameric IgM in the skin mucus. These results indicate that the skin pIgR transports tetrameric IgM into the skin mucus. The fugu pIgR exhibits a unique structure containing only two Ig-like domains corresponding to domain 1 and domain 4/5 of mammalian pIgR. This structure was sufficient for successful binding to tetrameric IgM. Teleost skin thus adopts the same Ig transport system as mammalian intestine via a unique pIgR.

Lymphocyte surface marker genes in fugu.

At present, much of the fish adaptive immune system remains unknown due to the paucity of marker-specific reagents (e.g. antibodies) to identify immune cells. The genomic sequence of Takifugu rubripes (fugu) represents an important resource to facilitate the identification of lymphocyte-related genes. Here, we review the recent works on B-lymphocyte markers, the heavy (H) chains of IgM and IgD, and Ig light chain, and T-cell marker gene homologues, CD3epsilon, CD3gamma/delta, CD4, and CD8alpha in a single fish species, fugu. Expressions of these B- and T-lymphocyte markers homologues in peripheral blood leukocytes and other lymphoid tissues of fugu suggest that these molecules in fish would be available as lymphocyte markers. These findings will lead us to develop reliable reagents for the identification of lymphocyte subpopulations. Fugu holds great promise as one of the model organisms for studies of development and evolution of adaptive and innate immunity in vertebrates.

Variable lymphocyte receptors in hagfish.

A previously uncharacterized type of variable lymphocyte receptors (VLR) was identified recently in the Sea lamprey. This jawless vertebrate generates an extensive VLR repertoire through differential insertion of neighboring diverse leucine-rich repeat (LRR) cassettes into an incomplete germ-line VLR gene. We report here VLR homologs from two additional lamprey species and the presence of two types of VLR genes in hagfish, the only other order of contemporary jawless vertebrates. As in the Sea lamprey, the incomplete hagfish germ-line VLR-A and -B genes are modified in lymphocyte-like cells to generate highly diverse repertoires of VLR-A and -B proteins via a presently undetermined mechanism. This jawless-fish mode of VLR diversification starkly contrasts with the rearrangement of Ig V(D)J gene segments used by all jawed vertebrates to produce diverse repertoires of T and B lymphocyte antigen receptors. The development of two very different strategies for receptor diversification at the dawn of vertebrate evolution approximately 500 million years ago attests to the fitness value of a lymphocyte-based system of anticipatory immunity.

Analysis and characterization of the expression of the secretory and membrane forms of IgM heavy chains in the pufferfish, Takifugu rubripes.

We investigated the structure and expression of immunoglobulin genes in the pufferfish, Takifugu rubripes, a highly prized and economically important fish species. The cDNA fragment that partially encodes the constant region of the IgM heavy chain was isolated in these animals by RACE using degenerate primers after which it was used as a probe for screening IgM heavy chains in a fugu splenic cDNA library. The structural feature of the constant region of fugu sIgM was found to consist of four constant domains (CH1 to CH4), while mIgM was shown to contain a deletion of the CH4 domain, and its transmembrane domain was directly spliced to the CH3 domain as found in other teleosts. This feature may be common to all teleosts. In addition, five VH genes isolated in this study fell into two families based on their variability. Analysis of genomic sequences from the fugu genomic database also showed that there are only two VH families in the genome. The IgM gene was preferentially expressed in presumptive lymphoid tissues. Moreover, in situ hybridization revealed that large numbers of IgM positive cells were widely distributed throughout the spleen, head kidney, kidney, and thymus, confirming that these tissues were major sites of antibody production in fish. The expressions of IgM in the mucosal organs such as the skin, gills, and intestine suggest that they, too, contribute to humoral immunity in aquatic animals. The expression of IgM mRNA in the early development stages of this fish suggests that its larval form possesses a protective defense mechanism against foreign invaders.

Fugu immunoglobulin D: a highly unusual gene with unprecedented duplications in its constant region.

We have isolated and characterized the cDNA that encodes IgD of fugu (Takifugu rubripes). Though the splicing of micro1 with the delta1 domain was similar to those reported for teleost IgDs, highly unusual and unprecedented domain duplications were found in the constant region of the fugu IgD. The structure of the fugu IgD is like VDJ-micro1-(delta1-delta2-delta3-delta4-delta5-delta6)(2)-delta7-deltam1-deltam2. Genomic sequence analysis of the fugu IgD gene supported the results of cDNA sequencing that the first six delta domains in the constant region are duplicated. Such a novel duplication pattern has not been reported in any other vertebrates. However, IgD secretory domains could not be identified in this study. The deduced amino acid sequence of the fugu IgD constant region showed high identity (35-55%) to the sequences of previously reported teleost IgDs. Gene expression analyses based on RT-PCR demonstrated that the IgD gene is preferentially expressed in presumptive lymphoid tissues; moreover, in situ hybridization showed that IgD-positive cells are distributed throughout the spleen and head kidney. The expression pattern is similar to that of IgM, corroborating the hypothesis that IgD plays an important role in the humoral immune system of this species.

In vitro effects of steroid hormones on IgM-secreting cells and IgM secretion in common carp (Cyprinus carpio).

The effects of steroid hormones on in vitro IgM-secreting cells (IgMSC) and IgM secretion by lymphocytes of the lymphoid organs in common carp, Cyprinus carpio were examined by ELISPOT and ELISA assay, respectively. Cells isolated from peripheral blood (PB), spleen and head kidney were cultured for 12, 24 and 48 h either in the absence or in the presence of steroid hormones, i.e. cortisol, testosterone, 11-ketotestosterone (11-KT), and estradiol-17beta (E(2)) at doses of 1, 10 and 100 ng/ml. Cortisol reduced the IgMSC numbers and IgM secretion by cells from all organs. In addition, cortisol induced apoptosis in lymphocytes from all organs. High dose of testosterone showed tissue-specific functions; it reduced the number of IgMSC and amount of IgM secretion by cells from spleen and head kidney, but not in PB, though IgM secretion was suppressed. However, no effects of sex steroids were observed in this study. The results show that sex-specific steroid hormones may have no immunosuppressive effects in common carp.

Characterization and expression of the immunoglobulin light chain in the fugu: evidence of a solitaire type.

In this study, we characterized the immunoglobulin light (IgL) chain gene and examined its expression in the fugu (Takifugu rubripes). The cDNA fragment that partially encodes the IgL chain was isolated by RACE and used as a probe for screening for IgL in a fugu splenic cDNA library. The IgL cDNA sequence that we found consisted of a variable (V(L)) and a constant (C(L)) segment. Its structural features were similar to the IgL isotype commonly found in teleosts. Genomic sequence analysis revealed that the IgL gene was organized as two V(L) gene segments (designed V(L1) and V(L2)) followed by single joining (J(L)) and C(L) segment. In addition, an unusual duplicate V(L1) gene segment was found downstream of the C(L) segment. The transcriptional orientation of the V(L) exons was found to be opposite to that of the J(L) and C(L) segments. Genomic blot hybridizations with V(L) and C(L) probes gave multibands, supporting the contention that the teleost IgL forms a multicluster. Both genomic and cDNA sequences analyses showed that all of the constant segments found in the fugu are identical, suggesting that no other isotypes could be found in this species. Comparison of the deduced amino acid sequence of the fugu C(L) domain with those of other species showed a high degree of identity (from 40 to 77%). IgL mRNAs were found to be expressed primarily in the lymphoid tissues. In situ hybridization revealed the presence of IgL-positive cells widely distributed throughout the spleen, head kidney, kidney, and thymus. These results support the contention that the lymphoid tissues are the major sites of antibody production in fish. Since IgL mRNA was also expressed in the skin and gill that are exposed to external antigens, it is likely that mucosal Ig plays an important role in immune protection.

A double staining flow cytometric assay for the detection of steroid induced apoptotic leucocytes in common carp (Cyprinus carpio).

Steroid hormones play an important role in the regulation of the immune system through different ways. In this in vitro study, the effects of steroid hormones on the apoptosis of leucocytes were evaluated to understand the involvement of this process in the immunocompetence of common carp. Prior to the investigation, a double staining flow cytometric assay using fluorescein diacetate (FDA), which reacts with esterases of viable cells, and propidium iodide (PI), an acid dye that binds with nuclear DNA, was established. FDA and PI negative cells were regarded as apoptotic. The FDA-PI technique is comparable to the Annexin V-PI technique and can be used in the quantification of the apoptosis of fish leucocytes accurately. The results suggest that the disappearance of esterases and externalization of phosphatidylserine (PS) may be common to many apoptotic pathways. Cells collected from peripheral blood, spleen, head kidney, and thymus were cultured for 16 h either in the absence or presence of steroid hormones, i.e. cortisol (F), testosterone, 11-ketotestosterone, and estradiol-17beta, and analyzed by flow cytometry followed by the FDA-PI method. Results showed that F induced apoptosis in leucocytes from blood and other lymphoid organs suggesting the role of F as an immune regulator. The participation of sex steroids to the immunocompetence of carp was not found, since they did not induce apoptosis of leucocytes in any organ.