Do ectothermic vertebrates have a home in which to affinity mature their antibody responses?

There has been a longstanding question of whether affinity maturation occurs in ectotherms, and if it does, where in tissues this happens. Although cold-blooded vertebrates (ectotherms) lack histologically discernible germinal centers, they have a fully functional Ig gene mutator enzyme (activation-induced cytidine deaminase: AID or Aicda). Protein and Ig cDNA transcript analyses provide evidence that ectotherms can, under certain conditions, demonstrate antibody affinity maturation, and somatic hypermutation of their Ig genes during secondary immune responses. Here, we review the evidence for antibody affinity maturation and somatic hypermutation of Ig V(D)J exons. We argue that past evidence of long-term intact antigen retention, and recent studies of in situ expression of AID transcripts, point to fish melanomacrophage clusters as sites functionally analogous to a germinal center. Recent work in zebrafish provides a way forward to test these predictions through V(D)J repertoire analyses on isolated, intact melanomacrophage clusters. This work has implications not only for vaccine use in aquaculture, but also for antibody affinity maturation processes in all ectothermic vertebrates.

Dual role of the Anopheles coluzzii Venus Kinase Receptor in both larval growth and immunity.

Vector-borne diseases and especially malaria are responsible for more than half million deaths annually. The increase of insecticide resistance in wild populations of Anopheles malaria vectors emphasises the need for novel vector control strategies as well as for identifying novel vector targets. Venus kinase receptors (VKRs) constitute a Receptor Tyrosine Kinase (RTK) family only found in invertebrates. In this study we functionally characterized Anopheles VKR in the Gambiae complex member, Anopheles coluzzii. Results showed that Anopheles VKR can be activated by L-amino acids, with L-arginine as the most potent agonist. VKR was not required for the fecundity of A. coluzzii, in contrast to reports from other insects, but VKR function is required in both Anopheles males and females for development of larval progeny. Anopheles VKR function is also required for protection against infection by Plasmodium parasites, thus identifying a novel linkage between reproduction and immunity in Anopheles. The insect specificity of VKRs as well as the essential function for reproduction and immunity suggest that Anopheles VKR could be a potentially druggable target for novel vector control strategies.

A Xenopus tadpole alternative model to study innate-like T cell-mediated anti-mycobacterial immunity.

Owing to the high incidence of multi-drug resistance and challenges posed by the complex and long duration of treatments, Mycobacterium tuberculosis (Mtb) infections remain a significant clinical burden, which would benefit from development of novel immuno-therapeutic-based treatment strategies. Among early immune effectors, invariant or innate-like (i)T cells are attracting attention because of their potential regulatory activity, which can shape anti-mycobacterial immune responses. Unlike conventional T cells, iT cells express a semi-invariant T cell receptor, and respond rapidly and robustly to molecular patterns presented by MHC class I-like molecules. To date, functional studies of iT cells in vivo has been problematic and the role of iT cells in anti-Mtb responses remains unclear. Here, after reviewing the recent literature on anti-mycobacterial iT cell immunity, we describe a novel alternative model system in the amphibian Xenopus laevis tadpoles during infection with Mycobacterium marinum (Mm). X. laevis tadpoles rely mostly on a few distinct prominent innate-like (i)T cell subsets, whose development and function are governed by distinct MHC class I-like molecules. Thus, X. laevis tadpoles provide a convenient and cost-effective in vivo model uniquely suited to investigate the roles of iT cells during mycobacterial infections. We have developed reverse genetics and MHC tetramer technology to characterize this MHC-like/iT system in tadpoles. Our study in X. laevis provides evidence of a conserved convergent function of iT cells in host defenses against mycobacteria between mammals and amphibians.

Flow Cytometric Analysis of Xenopus Immune Cells.

Flow cytometry is a versatile analytical platform capable of multiparameter analysis of more than a thousand individual cells per second. This technique is used to measure the physical and chemical characteristics of individual cells in a heterogeneous cell suspension as they pass through one or multiple lasers. Physical properties, such as size and internal complexity, are recorded as light scattering at different angles and are expressed as forward- and side-scatter, respectively. Following light excitation, fluorochromes conjugated to antibodies or intercalated with different cellular components reemit light at distinct wavelengths. This can identify a broad array of cell specific antigens, further defining distinct cell subsets based on activation, lineage, and developmental stage. The combination of labels that can be used depends on the laser used to excite the fluorochromes and on the detector and available antibodies. With the growing number of Xenopus-specific antibodies, flow cytometry can be used to identify, isolate, and characterize distinct immune cell subsets. In this protocol, different methods to obtain single-cell suspensions from various X. laevis tissues are described. A standard three-parameter procedure defining viability and two cell-surface markers is then described.

RNAi-Mediated Loss of Function of Xenopus Immune Genes by Transgenesis.

Generation of transgenic frogs through the stable integration of foreign DNA into the genome is well established in Xenopus This protocol describes the combination of transgenesis with stable RNA interference as an efficient reverse genetic approach to study gene function in Xenopus Initially developed in the fish medaka and later adapted to Xenopus, this transgenic method uses the I-SceI meganuclease, a "rare-cutter" endonuclease with an 18 bp recognition sequence. In this protocol, transgenic X. laevis with knocked down expression of a specific gene are generated using a double promoter expression cassette. This cassette, which is flanked by I-SceI recognition sites, contains the shRNA of choice under the control of the human U6 promoter and a green fluorescent protein (GFP) reporter gene under the control of the human EF-1α promoter. Prior to microinjection the plasmid is linearized by digestion with I-SceI and the entire reaction is then microinjected into one-cell stage eggs. The highly stringent recognition sequence of I-SceI is thought to maintain the linearized plasmid in a nonconcatamerized state, which promotes random integration of the plasmid transgene in the genome. The injected embryos are reared until larval stage 56 and then screened for GFP expression by fluorescence microscopy and assessed for effective knockdown by quantitative RT-PCR using a tail biopsy. Typically, the I-SceI meganuclease transgenesis technique results in 35%-50% transgenesis efficiency, a high survival rate (>35%) and bright nonmosaic GFP expression. A key advantage of this technique is that the high efficiency and nonmosaic transgene expression permit the direct use of F0 animals.

Functional characterization of the mucus barrier on the Xenopus tropicalis skin surface.

Mucosal surfaces represent critical routes for entry and exit of pathogens. As such, animals have evolved strategies to combat infection at these sites, in particular the production of mucus to prevent attachment and to promote subsequent movement of the mucus/microbe away from the underlying epithelial surface. Using biochemical, biophysical, and infection studies, we have investigated the host protective properties of the skin mucus barrier of the Xenopus tropicalis tadpole. Specifically, we have characterized the major structural component of the barrier and shown that it is a mucin glycoprotein (Otogelin-like or Otogl) with similar sequence, domain organization, and structural properties to human gel-forming mucins. This mucin forms the structural basis of a surface barrier (∼6 µm thick), which is depleted through knockdown of Otogl. Crucially, Otogl knockdown leads to susceptibility to infection by the opportunistic pathogen Aeromonas hydrophila To more accurately reflect its structure, tissue localization, and function, we have renamed Otogl as Xenopus Skin Mucin, or MucXS. Our findings characterize an accessible and tractable model system to define mucus barrier function and host-microbe interactions.

Exploring the functions of nonclassical MHC class Ib genes in Xenopus laevis by the CRISPR/Cas9 system.

A large family of highly related and clustered Xenopus nonclassical MHC class Ib (XNC) genes influences Xenopus laevis immunity and potentially other physiological functions. Using RNA interference (RNAi) technology, we previously demonstrated that one of XNC genes, XNC10.1, is critical for the development and function of a specialized innate T (iT) cell population. However, RNAi limitation such as a variable and unstable degree of gene silencing in F0 and F1 generations is hampering a thorough functional analysis of XNC10.1 and other XNC genes. To overcome this obstacle, we adapted the CRISPR/Cas9-mediated gene editing technique for XNC genes. We efficiently and specifically generated single gene knockouts of XNC10.1, XNC11, and XNC1 as well as double gene knockouts of XNC10.1 and XNC11 in X. laevis. In single XNC10.1 knockout X. laevis tadpoles, the absence of XNC10.1 and Vα6-Jα1.43 invariant T cell receptor rearrangement transcripts indicated XNC10.1 loss-of-function and deficiency in Vα6-Jα1.43 iT cells. Notably, targeting XNC10.1 did not affect neighboring XNC genes exhibiting high sequence similarity. Furthermore, XNC1 gene disruption induced mortality during developmental stage 47, suggesting some non-immune but essential function of this gene. These data demonstrate that the CRISPR/Cas9 system can be successfully adapted for genetic analysis in F0 generation of X. laevis.

Intronless and intron-containing type I IFN genes coexist in amphibian Xenopus tropicalis: Insights into the origin and evolution of type I IFNs in vertebrates.

Type I IFNs are considered to be the core IFN species in vertebrates because of their predominant antiviral effects. But, a puzzling question remains to be answered, as to how intronless type I IFN genes in amniotes might have evolved from intron-containing type I IFN genes in fish and amphibians. In this study, intronless and intron-containing type I IFNs were found in the amphibian model, Xenopus tropicalis, with a total of sixteen and five genes, respectively. The intronless IFNs can be divided into three subgroups, and the intron-containing ones into two subgroups, implying that a retroposition event might have occurred in amphibians, resulting in the generation of intronless type I IFN genes. Two models were tentatively proposed to explain the evolution of type I IFNs in vertebrates: in model A, fish should possess the most primitive multi-exon-containing type I IFNs, and intronless type I IFN genes in amphibians are the ancestor of modern intronless type I IFNs in amniotes; in model B, intronless type I IFN genes in X. tropicalis may just represent an independent bifurcation in this species or probably in amphibians, and intronless type I IFN genes in amniotes may have arisen from another retroposition event occurred in a transition period even when reptiles were diverged from amphibians. It is considered that the model B can reflect the current knowledge on the occurrence of intronless and intron-containing type I IFN genes in vertebrate lineages. This study thus contributes to a better understanding of the origin and evolution of type I IFNs in vertebrates, and of the occurrence of intronless I IFNs in higher vertebrates.

The repetitive portion of the Xenopus IgH Mu switch region mediates orientation-dependent class switch recombination.

Vertebrates developed immunoglobulin heavy chain (IgH) class switch recombination (CSR) to express different IgH constant regions. Most double-strand breaks for Ig CSR occur within the repetitive portion of the switch regions located upstream of each set of constant domain exons for the Igγ, Igα or Igϵ heavy chain. Unlike mammalian switch regions, Xenopus switch regions do not have a high G-density on the non-template DNA strand. In previous studies, when Xenopus Sµ DNA was moved to the genome of mice, it is able to support substantial CSR when it is used to replace the murine Sγ1 region. Here, we tested both the 2kb repetitive portion and the 4.6 kb full-length portions of the Xenopus Sµ in both their natural (forward) orientation relative to the constant domain exons, as well as the opposite (reverse) orientation. Consistent with previous work, we find that the 4.6 kb full-length Sµ mediates similar levels of CSR in both the forward and reverse orientations. Whereas, the forward orientation of the 2kb portion can restore the majority of the CSR level of the 4.6 kb full-length Sµ, the reverse orientation poorly supports R-looping and no CSR. The forward orientation of the 2kb repetitive portion has more GG dinucleotides on the non-template strand than the reverse orientation. The correlation of R-loop formation with CSR efficiency, as demonstrated in the 2kb repetitive fragment of the Xenopus switch region, confirms a role played by R-looping in CSR that appears to be conserved through evolution.

Semi-solid tumor model in Xenopus laevis/gilli cloned tadpoles for intravital study of neovascularization, immune cells and melanophore infiltration.

Tumors have the ability to grow as a self-sustaining entity within the body. This autonomy is in part accomplished by the tumor cells ability to induce the formation of new blood vessels (angiogenesis) and by controlling cell trafficking inside the tumor mass. These abilities greatly reduce the efficacy of many cancer therapies and pose challenges for the development of more effective cancer treatments. Hence, there is a need for animal models suitable for direct microscopy observation of blood vessel formation and cell trafficking, especially during early stages of tumor establishment. Here, we have developed a reliable and cost effective tumor model system in tadpoles of the amphibian Xenopus laevis. Tadpoles are ideally suited for direct microscopy observation because of their small size and transparency. Using the thymic lymphoid tumor line 15/0 derived from, and transplantable into, the X. laevis/gilli isogenic clone LG-15, we have adapted a system that consists in transplanting 15/0 tumor cells embedded into rat collagen under the dorsal skin of LG-15 tadpole recipients. This system recapitulates many facets of mammalian tumorigenesis and permits real time visualization of the active formation of the tumor microenvironment induced by 15/0 tumor cells including neovascularization, collagen rearrangements as well as infiltration of immune cells and melanophores.

Xenbase, the Xenopus model organism database; new virtualized system, data types and genomes.

Xenbase (http://www.xenbase.org), the Xenopus frog model organism database, integrates a wide variety of data from this biomedical model genus. Two closely related species are represented: the allotetraploid Xenopus laevis that is widely used for microinjection and tissue explant-based protocols, and the diploid Xenopus tropicalis which is used for genetics and gene targeting. The two species are extremely similar and protocols, reagents and results from each species are often interchangeable. Xenbase imports, indexes, curates and manages data from both species; all of which are mapped via unique IDs and can be queried in either a species-specific or species agnostic manner. All our services have now migrated to a private cloud to achieve better performance and reliability. We have added new content, including providing full support for morpholino reagents, used to inhibit mRNA translation or splicing and binding to regulatory microRNAs. New genomes assembled by the JGI for both species and are displayed in Gbrowse and are also available for searches using BLAST. Researchers can easily navigate from genome content to gene page reports, literature, experimental reagents and many other features using hyperlinks. Xenbase has also greatly expanded image content for figures published in papers describing Xenopus research via PubMedCentral.

Expression cloning of camelid nanobodies specific for Xenopus embryonic antigens.

Developmental biology relies heavily on the use of conventional antibodies, but their production and maintenance involves significant effort. Here we use an expression cloning approach to identify variable regions of llama single domain antibodies (known as nanobodies), which recognize specific embryonic antigens. A nanobody cDNA library was prepared from lymphocytes of a llama immunized with Xenopus embryo lysates. Pools of bacterially expressed cDNAs were sib-selected for the ability to produce specific staining patterns in gastrula embryos. Three different nanobodies were isolated: NbP1 and NbP3 stained yolk granules, while the reactivity of NbP7 was predominantly restricted to the cytoplasm and the cortex. The isolated nanobodies recognized specific protein bands in immunoblot analysis. A reverse proteomic approach identified NbP1 target antigen as EP45/Seryp, a serine protease inhibitor. Given the unique stability of nanobodies and the ease of their expression in diverse systems, we propose that nanobody cDNA libraries represent a promising resource for molecular markers for developmental biology.

Unusual evolutionary conservation and further species-specific adaptations of a large family of nonclassical MHC class Ib genes across different degrees of genome ploidy in the amphibian subfamily Xenopodinae.

Nonclassical MHC class Ib (class Ib) genes are a family of highly diverse and rapidly evolving genes wherein gene numbers, organization, and expression markedly differ even among closely related species rendering class Ib phylogeny difficult to establish. Whereas among mammals there are few unambiguous class Ib gene orthologs, different amphibian species belonging to the anuran subfamily Xenopodinae exhibit an unusually high degree of conservation among multiple class Ib gene lineages. Comparative genomic analysis of class Ib gene loci of two divergent (~65 million years) Xenopodinae subfamily members Xenopus laevis (allotetraploid) and Xenopus tropicalis (diploid) shows that both species possess a large cluster of class Ib genes denoted as Xenopus/Silurana nonclassical (XNC/SNC). Our study reveals two distinct phylogenetic patterns among these genes: some gene lineages display a high degree of flexibility, as demonstrated by species-specific expansion and contractions, whereas other class Ib gene lineages have been maintained as monogenic subfamilies with very few changes in their nucleotide sequence across divergent species. In this second category, we further investigated the XNC/SNC10 gene lineage that in X. laevis is required for the development of a distinct semi-invariant T cell population. We report compelling evidence of the remarkable high degree of conservation of this gene lineage that is present in all 12 species of the Xenopodinae examined, including species with different degrees of ploidy ranging from 2, 4, 8 to 12 N. This suggests that the critical role of XNC10 during early T cell development is conserved in amphibians.

A secretory cell type develops alongside multiciliated cells, ionocytes and goblet cells, and provides a protective, anti-infective function in the frog embryonic mucociliary epidermis.

The larval epidermis of Xenopus is a bilayered epithelium, which is an excellent model system for the study of the development and function of mucosal and mucociliary epithelia. Goblet cells develop in the outer layer while multiciliated cells and ionocytes sequentially intercalate from the inner to the outer layer. Here, we identify and characterise a fourth cell type, the small secretory cell (SSC). We show that the development of these cells is controlled by the transcription factor Foxa1 and that they intercalate into the outer layer of the epidermis relatively late, at the same time as embryonic hatching. Ultrastructural and molecular characterisation shows that these cells have an abundance of large apical secretory vesicles, which contain highly glycosylated material, positive for binding of the lectin, peanut agglutinin, and an antibody to the carbohydrate epitope, HNK-1. By specifically depleting SSCs, we show that these cells are crucial for protecting the embryo against bacterial infection. Mass spectrometry studies show that SSCs secrete a glycoprotein similar to Otogelin, which may form the structural component of a mucus-like protective layer, over the surface of the embryo, and several potential antimicrobial substances. Our study completes the characterisation of all the epidermal cell types in the early tadpole epidermis and reinforces the suitability of this system for the in vivo study of complex epithelia, including investigation of innate immune defences.

Nonclassical MHC class I-dependent invariant T cells are evolutionarily conserved and prominent from early development in amphibians.

Human and murine MHC nonclassical class Ib-restricted invariant T (iT) cell subsets, such as invariant natural killer T cells (iNKT) and mucosal-associated invariant T cells, have specialized functions early in immune responses, especially in modulating subsequent adaptive immune responses. Here, we characterize a prominent iT population in the amphibian Xenopus laevis and show the requirement of the class Ib molecule, Xenopus nonclassical gene 10, in its differentiation and function. Using Xenopus nonclassical gene 10 tetramers and RNAi loss of function by transgenesis, we identified a large class Ib-dependent CD8(-)/CD4(-) iT subset in unmanipulated frogs and tadpoles. This population is critical for antiviral immunity during early larval stages when classical MHC class Ia function is suboptimal. Furthermore, in young tadpoles with low class Ia expression, deep sequencing revealed additional preponderant invariant T cell receptor (TCR)α rearrangements, implying other iT cell subsets and a predominant selection process mediated by other class Ib molecules. The restriction and requirement of class Ib molecules for development and antiviral immunity of a mammalian iNKT or mucosal-associated invariant T cell counterpart in the amphibian Xenopus show the importance of iT cells in the emergence and evolution of the adaptive immune system.

Only skin deep: shared genetic response to the deadly chytrid fungus in susceptible frog species.

Amphibian populations around the world are threatened by an emerging infectious pathogen, the chytrid fungus Batrachochytrium dendrobatidis (Bd). How can a fungal skin infection kill such a broad range of amphibian hosts? And do different host species have a similar response to Bd infection? Here, we use a genomics approach to understand the genetic response of multiple susceptible frog species to Bd infection. We characterize the transcriptomes of two closely related endangered frog species (Rana muscosa and Rana sierrae) and analyse whole genome expression profiles from frogs in controlled Bd infection experiments. We integrate the Rana results with a comparable data set from a more distantly related susceptible species (Silurana tropicalis). We demonstrate that Bd-infected frogs show massive disruption of skin function and show no evidence of a robust immune response. The genetic response to infection is shared across the focal susceptible species, suggesting a common effect of Bd on susceptible frogs.

Expansion and diversification of the signaling capabilities of the CD2/SLAM family in Xenopodinae amphibians.

We studied the evolution of the CD2 family in tetrapods by extracting and analyzing CD2-like genes from the genome of the amphibian species Silurana (Xenopus) tropicalis. An exhaustive analysis of the genomic and cDNA databases resulted in the identification of at least 70 CD2-like genes. The predicted receptors mostly maintain the typical VC2 ectodomains, but are highly diverse in their C-termini, which suggests a broad range of signaling capacities. Apart from the presumed monomeric receptors with ITSM and/or ITIM motifs, the Silurana family includes secreted proteins. Furthermore, a fraction of the receptors contain a conserved TM subtype with the NxxR motif that is known to promote an association with the FcRγ subunit and that was previously found in the members of the FcR- and KIR-related receptors. The expression analysis of a sample of the genes showed broad tissue distribution and gene-specific expression patterns. Phylogenetic analysis predicted that the CD58, CD150/SLAM, and SLAMF8 genes were maintained as single-copy genes in both mammals and amphibians, while others expanded/contracted in a lineage-specific manner.

Phylogenetic and developmental study of CD4, CD8 α and ß T cell co-receptor homologs in two amphibian species, Xenopus tropicalis and Xenopus laevis.

CD4 and CD8 co-receptors play critical roles in T cell development and activation by interacting both with T cell receptors and MHC molecules. Although homologs of these genes have been identified in many jawed vertebrates, there are still unresolved gaps concerning their evolution and specialization in MHC interaction and T cell function. Using experimental and computational procedures we identified CD4, CD8α and CD8ß gene homologs both in Xenopus tropicalis, whose full genome has been sequenced, and its sister species Xenopus laevis. Multiple alignments of deduced amino acid sequences reveal a poor conservation of the residues involved in binding of CD4 to MHC class II, and CD8α to class I in non-mammalian species, presumably related to the co-evolutionary pressure of MHC I and II genes. Phylogenetic study suggests that Xenopodinae co-receptor genes are more closely related to their homologs in other tetrapods than those of bony fish. Furthermore, the developmental and cell-specific expression patterns of these genes in X. laevis are very similar to that of mammals. X. laevis CD4 is mainly expressed by peripheral non-CD8 T cells and detected in the thymus as early as four days post-fertilization (dpf) at the onset of thymic organogenesis. CD8ß expression is specific to adult surface CD8(+) T cells and thymocytes, and is first detected in the thymus at 5 dpf in parallel with productive TCRγ transrcipts, whereas productive TCRß and α rearrangements are not detected before 7-9 dpf.

The amphibians Xenopus laevis and Silurana tropicalis possess a family of activating KIR-related Immunoglobulin-like receptors.

In this study, we searched the amphibian species Xenopus laevis and Silurana (Xenopus) tropicalis for the presence of genes homologous to mammalian KIRs and avian CHIRs (KRIR family). By experimental and computational procedures, we identified four related ILR (Ig-like Receptors) genes in S. tropicalis and three in X. laevis. ILRs encode type I transmembrane receptors with 3-4 Ig-like extracellular domains. All predicted ILR proteins appear to be activating receptors. ILRs have a broad expression pattern, the gene transcripts were found in both lymphoid and non-lymphoid tissues. Phylogenetic analysis shows that the amphibian KRIR family receptors evolved independently from their mammalian and avian counterparts. The only conserved structural element of tetrapod KRIRs is the NxxR motif-containing transmembrane domain that facilitates association with FcRgamma subunit. Our findings suggest that if KRIRs of various vertebrates have any common function at all, such a function is activating rather than inhibitory.

Expression of immune genes during metamorphosis of Xenopus: a survey.

This review focuses on what is known about the immune transcriptome during metamorphosis of Xenopus laevis and Silurana (Xenopus) tropicalis. This subject is of importance to obtain a global understanding of the physiological changes operating during metamorphosis. In turn, a good knowledge of the physiology of amphibian metamorphosis may contribute to the fight against amphibian decline and help the development of alternative toxicologic assays. By examining what is known on the expression of innate and adaptive immune genes during metamorphosis, it becomes clear that our knowledge of the anatomy of the tadpole "immunome" is fragmentary. Since a wealth of data sits in cDNA sequences, I am making a first attempt to enrich our knowledge on this subject. I exemplify that mining EST data can rapidly provide us with the necessary tools to unravel the cross-talk between thyroid hormone signalling during metamorphosis and larval immune system changes.