Inducible hkRASG12D Expression in Zebrafish Hematopoietic Cells Leads to Myeloproliferative Disease.

Activating mutations in RAS family members are common in myeloproliferative disease (MPD) and acute myeloid leukemia (AML). Because the zebrafish has proven to be an excellent leukemia model and can be used in forward genetic and chemical screens to identify modulators of disease pathways, we developed a transgenic zebrafish model of RAS-induced myeloproliferative disease. Stable transgenic zebrafish lines were created in which the ubiquitous B-actin promoter drives expression of a loxed GFP transgene. Upon CRE-mediated recombination, the loxed GFP cassette is excised and the human kRASG12D transgene is expressed. When mated to hsp70-CRE transgenic zebrafish that express CRE recombinase when the animals are heated to 37C for 1 hour, a cohort of 300 double transgenic embryos was generated. In the heat-shocked group, the medial survival time was 25 days, suggesting that oncogenic RAS expression is lethal in developing zebrafish larvae. Of those animals that survived past this time window, most developed sarcomatous muscle tumors. In contrast, most of the double transgenic zebrafish that did not receive heatshock survived to adulthood. Because the hsp70 promoter is active in development and can be induced by stress, the hsp70-CRE transgenic zebrafish line exhibits low levels of CRE expression even in the absence of heatshock. In the non-heat shocked cohort, 10 of 120 double transgenic zebrafish developed MPD by 66 days of life. Histological examination and fluorescence cytometry analysis revealed an expansion of myeloid cell populations within the kidney, comprising granulocytic and monocytic cells in various stages of differentiation, mimicking myeloproliferative diseases seen in both human and mouse. To specifically induce kRASG12D expression in hematopoietic cells, kidneys were dissected from healthy double transgenic animals; heat shocked ex vivo, and then transplanted into sub-lethally irradiated recipient fish. Upon analysis of transplant animals at three months of age, flow cytometry confirmed that MPD had developed in the recipient fish, however other tumor subtypes were not observed. When kidney marrow from transplant animals having MPD were introduced into sub-lethally irradiated secondary recipients, transplant efficiency was greatly reduced. Taken together these results suggest that, as is seen in MPD in mammals, myeloid cells are not fully transformed in our model. In summary, we show that inducible hkRASG12D expression in zebrafish hematopoietic cells leads to myeloproliferative disease and suggest that this model will allow for the identification of novel pathways responsible for full transformation leading to AML.