Diversity of Class I Major Histocompatibility Genes From Divergent Haplotypes in Zebrafish

Abstract 1058

The zebrafish is an important animal model for stem cell biology, cancer, and immunology research. Histocompatibility represents a key intersection of these disciplines, particularly in the context of transplantation experiments that distinguish between autologous and allogeneic tissues. Major histocompatibility (MH) genes are considered the most polymorphic genes in vertebrates, yet this immense variation occurs while maintaining conserved roles in antigen presentation. Histocompatibility in zebrafish remains poorly understood, requiring the identification of the classical MH genes as well as an evaluation of the variation between haplotypes. Although at least 11 putative zebrafish Class I MH U lineage genes have been isolated from cDNA libraries, their genomic organization and haplotype assignments remain uncharacterized for the majority.

We focused our study on a set of diverse zebrafish Class I MH genes that segregate with specific haplotypes at chromosome 19, and for which donor-recipient matching was previously shown to be associated with improved engraftment after transplantation. Inside the conserved psmb8 and tpsn flanking gene regions on chromosome 19, Class I MH haplotypes can differ markedly among zebrafish strains including Tubingen and AB. Interestingly, the distinct haplotypes at chromosome 19 appear to maintain non-overlapping sets of genes, and also have gene copy number differences. For example, haplotype A contains the genes mhc1uda, mhc1uea, and mhc1ufa, with tap2 genes located in between each set of MH genes. In contrast, haplotype B contains mhc1uba and mhc1uca genes that are separated instead by a tapbp gene.

Among these two highly divergent haplotypes, mhc1uea and mhc1uda from haplotype A appear to be more closely related to one another than to either of the MH genes on haplotype B. Similarly, the mhc1uba and mhc1uca genes from haplotype B share higher levels of sequence identity than in comparison with genes from haplotype A. These findings differ markedly from the MH genes in mammals where the highest similarity is between alleles of the same Class I gene (for example, HLA-A in humans), instead of the other genes on the same haplotype (eg. HLA-B and HLA-C). In addition, the zebrafish MH gene sequences are very closely related to MH genes from other fish species. As an example, mhc1ufa is more similar to a Class I MH gene from gibuna carp than to other zebrafish MH genes. These data in zebrafish indicate that selected Class I MH gene polymorphisms have been preserved within the species since before the species diverged from other teleost fishes, suggesting a selective advantage for this unique form of MH diversity. To determine mRNA expression levels of the Class I MH genes, quantitative PCR was performed on liver, spleen, kidney marrow, testis, gill, intestine and heart from zebrafish that were homozygous for either haplotype A or haplotype B. Within haplotype A, expression of mhc1uda, mhc1uea and mhc1ufa is relatively equivalent, with mhc1ufa expressed at the highest levels overall. For haplotype B, mhc1uba has the highest levels in all tissues examined, while mhc1uca expression is 10–100 fold lower and somewhat variable between tissues. Putative peptide anchor residues are highly conserved between species. Those anchor residues that are conserved in all species including mammals, as well as two additional residues that are conserved in teleosts, are also all conserved in mhc1uda, mhc1uba, and mhc1uca. In contrast, mhc1uea and mhce1ufa each have single amino acid substitutions at critical residues, K146N and Y59F, respectively. These substitutions do not necessarily disqualify these molecules from consideration as classical MH Class I genes.

In conclusion, we propose that mhc1uba and mhc1uda function as classical MH Class I genes within divergent haplotypes, based on mRNA expression levels and tissue distribution, as well as sequence properties including conservation of putative peptide anchor residues. Using these same criteria, additional zebrafish Class I MH genes may be identified to serve similar roles. These predictions require verification via additional functional transplantation experiments that are currently underway in our lab. Defining the functional zebrafish Class I MH genes will provide an important foundation for future studies in immunology and transplantation.