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A comparative study of collagen IV basement membranes in shark, hagfish, frog, and salamander. 

Xenopus is a genus of African clawed frogs including two species, X. tropicalis and X. laevis that are extensively used in experimental biology, immunology, and biomedical studies. The availability of fully sequenced and annotated Xenopus genomes is strengthening genome-wide analyses of gene families and transgenesis to model human diseases. However, inaccuracies in genome annotation for genes involved in the immune system (i.e., immunome) hamper immunogenetic studies. Furthermore, advanced genome technologies (e.g., single-cell and RNA-Seq) rely on well-annotated genomes. The annotation problems of Xenopus immunome include a lack of established orthology across taxa, merged gene models, poor representation in gene pages on Xenbase, misannotated genes and missing gene IDs. The Xenopus Research Resource for Immunobiology in collaboration with Xenbase and a group of investigators are working to resolve these issues in the latest versions of genome browsers. In this review, we summarize the current problems of previously misannotated gene families that we have recently resolved. We also highlight the expansion, contraction, and diversification of previously misannotated gene families. 

Natural killer (NK) cells play major roles in innate immunity against viruses and cancer. Natural killer receptors (NKR) expressed by NK cells recognize foreign- or self-ligands on infected and transformed cells as well as healthy cells. NKR genes are the most rapidly evolving loci in vertebrates, and it is generally difficult to detect orthologues in different taxa. The unique exception is NKp30, an activating NKR in mammals that binds to the self-ligand B7H6. The NKp30-encoding gene, NCR3, has been found in most vertebrates including sharks, the oldest vertebrates with human-type adaptive immunity. NCR3 has a special, non-rearranging VJ-type immunoglobulin superfamily (IgSF) domain that predates the emergence of the rearranging antigen receptors. Herein we show that NCR3 loci are linked to the shark major histocompatibility complex (MHC), proving NCR3’s primordial association with the MHC. We identified eight subtypes of differentially expressed highly divergent shark NCR3 family genes. Using in situ hybridization, we detected one subtype, NS344823, to be expressed by predominantly single cells outside of splenic B cell zones. The expression by non-B cells was also confirmed by PCR in peripheral blood lymphocytes. Surprisingly, high expression of NS344823 was detected in the thymic cortex, demonstrating NS344823 expression in developing T cells. Finally, we show for the first time that shark T cells are found as single cells or in small clusters in the splenic red pulp, also unassociated with the large B cell follicles we previously identified. 

Since the discovery of the T cell receptor (TcR), immunologists have assigned somatic hypermutation (SHM) as a mechanism employed solely by B cells to diversify their antigen receptors. Remarkably, we found SHM acting in the thymus on α chain locus of shark TcR. SHM in developing shark T cells likely is catalyzed by activation-induced cytidine deaminase (AID) and results in both point and tandem mutations that accumulate non-conservative amino acid replacements within complementarity-determining regions (CDRs). Mutation frequency at TcRα was as high as that seen at B cell receptor loci (BcR) in sharks and mammals, and the mechanism of SHM shares unique characteristics first detected at shark BcR loci. Additionally, fluorescence in situ hybridization showed the strongest AID expression in thymic corticomedullary junction and medulla. We suggest that TcRα utilizes SHM to broaden diversification of the primary αβ T cell repertoire in sharks, the first reported use in vertebrates.