Clonal analysis of native and malignant hematopoiesis using color barcoding in zebrafish

Abstract

Hematopoietic stem cells (HSCs) emerge from the dorsal aorta during embryogenesis and sustain lifelong blood production. Over time, HSC clones accumulate mutations that may cause clonal expansion at the expense of normal clones, a state preceding hematopoietic malignancies such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Assessing HSC clone number and contribution to mature blood in both native and malignant settings has remained challenging. It is also unclear which combinations of mutations promote or cooperate in clonal expansion in vivo. Here, I developed a clonal marking strategy in which each HSC is labeled with a unique and heritable fluorescent hue using the transgenic line zebrabow. By crossing stable zebrafish lines, I paired zebrabow with a tamoxifen-inducible cre recombinase driven by draculin regulatory elements, which are active in embryonic HSCs, to induce clonal labeling during embryogenesis. I clustered populations of mature granulocytes with unique fluorescent hues in adulthood and from that calculated the number of HSC clones born during development. I discovered that 21 HSC clones on average exist prior to HSC emergence from the dorsal aorta and 30 clones are present during peak production. By longitudinal analysis, I found that these clones stably contribute to hematopoiesis for over one year. This result, paired with near complete labeling of the hematopoietic system, suggests all embryonic HSCs participate in forming the blood system. I also introduced combinations of mutations found in human MDS/AML into zebrabow embryos to prospectively determine which mutations promote clonal expansion. Mosaic mutations in epigenetic factors — asxl1, ezh2, dnmt3a, dnmt3b, tet2, and others — resulted in measureable expansion of single color clones. I found the majority of zebrafish exhibiting clonal expansion harbored frame-shift mutations in asxl1 with >25% variant allele frequency. The clonal expansion remained non-pathological up to 26 months, consistent with a pre-malignant state. When I paired epigenetic mutations with over-expression of the oncogenes Jak2V617F or FLT3-ITD, I observed clonal expansion and myeloid malignancy by 2-6 months post-fertilization. My findings provide the first quantitative insights into early clonal events that regulate blood development, and they lay the groundwork for a mutational ‘code’ that coordinates clonal expansion.