Development of a Limiting Dilution Assay and MHC-Matched Transplantation Model to Detect Hematopoietic Stem Cell Activity in Zebrafish

The zebrafish is a powerful model for the discovery of novel genetic mechanisms through large scale forward and reverse screening methodologies, and has been used prominently for the study of hematopoiesis. Methodologies capable of assessing hematopoietic stem cell (HSC) activity, however, are largely undeveloped. Here, we created a long-term limit dilution hematopoietic reconstitution assay in adult zebrafish, defined zebrafish haplotypes at the proposed core Major Histocompatibility Complex (MHC) locus, and tested their functional significance by performing the first matched and unmatched transplants. We identified a sublethal radiation dose of 25Gy which was optimal for hematopoietic reconstitution while minimizing mortality presumably due to radiation damage of other organs. Our method detects multi-lineage repopulation in primary and secondary recipients. Using Poisson statistics, our limit dilution analyses suggest that at least 1 in 65,000 nucleated marrow cells in zebrafish contain repopulating activity, consistent with mammalian marrow HSC frequency. Finally, we characterized the genes in the proposed MHC core locus on chromosome 19 for one family used for sibling marrow transplantations. We were able to identify the four parental MHC haplotypes by sequencing PCR products amplified for specific MHC Class I genes: mhc1uea, mhc1ufa, mhc1uda, mhc1uba, mhc1uca, loc751750, and mhc1uxa2. F1 generation siblings were subsequently typed, and we demonstrated that matching the donor and recipient MHC haplotypes at this chromosome 19 locus dramatically increases engraftment and percentage of donor chimerism in recipients compared to MHC-mismatched donors and recipients. At 15 weeks post-transplant, MHC-matched recipients showed engraftment in 4 of 5 surviving fish with mean donor chimerism of 59.15% +/− 25.1% for myeloid cells and 8.26% +/− 6.2% for lymphoid cells. Those animals receiving MHC-mismatched donor marrow had only 3.92% +/− 2.1% myeloid donor chimerism and 0.97% +/− 0.5% lymphoid donor chimerism. These data represent the first assay allowing long term HSCs to be distinguished from other hematopoietic progenitor fates and provides the first functional test of MHC genes between zebrafish haplotypes. This method opens the door to MHC-matched long-term transplantation experiments in zebrafish that have previously not been possible, including competitive transplantation experiments with zebrafish mutants already identified in prior genetic screens, and long-term tumor transplantation assays. By harnessing the unique genetic and screening advantages of the zebrafish model, such experiments may provide critical insight into mammalian transplantation biology.