Visualizing Clonal Hematopoiesis Associated with gata2 Deficiency in Zebrafish Using Color-Barcoding

Myeloid neoplasms, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), arise from abnormal hematopoietic stem and progenitor cells (HSPCs) with acquired somatic mutations. Germline GATA2 haploinsufficiency is associated with an autosomal dominant familial MDS/AML syndrome. Leukemic transformation in GATA2 haploinsufficiency occurs with acquisition of secondary mutations or cytogenetic abnormalities. We generated gata2 heterozygous zebrafish to model this germline predisoposition syndrome, and evaluate clonal architecture and dynamics over time at steady-state and in the presence of additional mutations. We hypothesized that clinically benign clonal hematopoiesis precedes the establishment of myeloid neoplasms in GATA2 haploinsufficiency. We used a lineage tracing system of zebrafish Brainbow, Zebrabow, to track the hematopoietic output of individually colored HSPC clones, with the goal to detect abnormal expansion of single color clones in the early stages of leukemogenesis, indicating clonal hematopoiesis. A heterozygous line of GATA2 zebrafish ortholog gata2b was generated in the Zebrabow background. We introduced combinations of genetic mutations described in GATA2 associated MDS and AML into Zebrabow embryos with CRISPR/Cas9 technology. Zebrafish embryos, either wild-type or heterozygous for gata2b, were injected with Cas9 protein and guide RNAs targeting zebrafish orthologs of ASXL1 and STAG2. Zebrabow color recombination was induced to color-label stem cells prior to their emergence from the dorsal aorta. At this time roughly 20 stem cell clones exist in the zebrafish embryo. The animals were then raised to adulthood. Kidney marrow cells of juvenile zebrafish were analyzed at various time points in between 2-4 months post-fertilization (mpf) by flow cytometry and morphology on cytospins. At steady-state, juvenile gata2b^+/- zebrafish had a decreased myeloid compartment in the kidney marrow. A third of gata2b^+/- zebrafish also had an expansion of single color clones in the kidney marrow by 3 mpf, contributing to ≥30% of granulocytes, while no wild-type clutchmate demonstrated single color expansion to this degree at steady-state. Mosaic combinations of mutations in the presence of gata2b deficiency resulted in an increase of precursor and a more pronounced decrease of myeloid populations in the kidney marrow at 3 mpf, compared to wild-type zebrafish injected with same mutations. No changes were observed in the lymphoid compartment. Single color expansion was observed in gata2b^+/- zebrafish three times more frequently than in wild-type zebrafish. In summary, gata2b deficiency sensitizes juvenile zebrafish to developing myelocytopenia at steady-state, reminiscent of well-characterized monocytopenia in GATA2 deficiency. We found that myelocytopenia is more profound and associated with an increase in early precursors upon additional somatic mutations associated with MDS/AML, possibly due to a differentiation block in myelopoiesis. Single color dominance observed in the hematopoeitic compartment of juvenile gata2b^+/- zebrafish using the Zebrabow color-barcoding system presents a potential mechanism underlying the predisposition to myeloid neoplasms associated with GATA2 deficiency, and provides a tool for visualizing asymptomatic premalignant state in disease pathogenesis.