Distinct Sets Of Class I Major Histocompatibility Genes In Zebrafish Expressed From Six Divergent Haplotypes

The zebrafish offers unique strengths as an animal model for stem cell biology, cancer, and immunology research. A better understanding of zebrafish histocompatibility genes will further strengthen this foundation. While histocompatibility genes are recognized as the most polymorphic genes in the mammalian genome, by comparison the sequence diversity among Class I genes in teleost species (bony fish) is substantially greater. The functional consequences of this high diversity are not yet well understood. We focused our study on zebrafish Class I major histocompatibility (MH) genes that segregate with specific haplotypes at chromosome 19, as donor-recipient matching at this locus has been shown to improve engraftment after hematopoietic transplantation. Interestingly, these haplotypes display marked differences in genomic sequence at this locus including variable gene copy number and content.

We examined six haplotypes that each display unique patterns of single nucleotide polymorphisms (SNPs) in linked genes psmb8 and zbtb22 that flank the Class I major histocompatibility gene locus on chromosome 19. We identified ten full-length Class I genes that are expressed in zebrafish and assigned each of the divergent genes to at least one haplotype. Genomic Southern blot data confirmed the presence of one to three unique Class I MH genes per haplotype. Sequence examination of these ten genes revealed expected motifs for Class I function, including predicted interaction residues for CD8 and beta-2-microglobulin. However, the predicted peptide anchor residues for some of the genes displayed occasional substitutions. This suggests possible flexibility in antigen binding among these genes that may influence their functional repertoire.

Using qPCR, we showed non-overlapping and sometimes co-dominant expression patterns of these genes that were specific to fish homozygous for each particular haplotype. For example, mhc1uda, mhc1uea, mhc1ufa were expressed at relatively equivalent levels from haplotype A. In contrast, from haplotype B one of the two genes (mhc1uca) was expressed at levels approximately ten fold lower than the other gene associated with that haplotype (mhc1uba). Similarly, for haplotype C mhc1uka was expressed at levels much lower than mhc1uja. Interestingly, mhc1uca and mhc1uka have relatively atypical sequences among these genes including the presence of additional exons which may indicate that they function in a non-classical capacity. For haplotype D, mhc1uga was the only gene found to be expressed by that haplotype. For haplotype E, only mhc1uia was found to be expressed but genomic Southern blot analysis indicated the presence of an additional gene with unknown sequence within this haplotype. For haplotype F only mhc1uha was expressed.

From these data we can predict that most of these unique zebrafish Class I genes function either co-dominantly or as the exclusive classical genes within their respective haplotypes. These predictions will require functional verification, such as via labeled tumor transplantation experiments that are currently underway in our laboratory. Dissecting the function and genetic diversity of zebrafish MHC genes promises to advance future studies with this important animal model including transplantation experiments for disease modeling of cancer, graft versus host disease, and immune response to infectious diseases.