A novel zebrafish jak2a(V581F) model shared features of human JAK2(V617F) polycythemia vera

OBJECTIVE: The Janus kinase 2 (JAK2) is important for embryonic primitive hematopoiesis. A gain-of-function JAK2 (JAK2(V617F)) mutation in human is pathogenetically linked to polycythemia vera (PV). In this study, we generated a zebrafish orthologue of human JAK2(V617F) (referred herewith jak2a(V581F)) by site-directed mutagenesis and examined its relevance as a model of human PV. METHODS: Zebrafish embryos at 1-cell stage were injected with jak2a(V581F) mRNA (200pg/embryos). In some experiments, the embryos were treated with a specific JAK2 inhibitor TG101209. The effects of jak2a stimulation on hematopoiesis, jak/stat signaling and erythropoietin signaling were evaluated at 18hours-post-fertilization (hpf). RESULTS: Injection with jak2a(V581F) mRNA significantly increased erythropoiesis, as enumerated by flow cytometry based on gata1(+) population in dissociated Tg(gata1:gfp) embryos. The response was reduced by stat5.1 morpholino co-injection (Control: 4.37+/-0.08%; jak2a(V581F) injected: 5.71+/-0.07%, Co-injecting jak2a(V581F) and stat5.1 morpholino: 4.66+/-0.13%, p<0.01). jak2a(V581F) mRNA also up-regulated gata1 (1.83+/-0.08 fold, p=0.005), embryonic alpha (alphaeHb: 1.61+/-0.12 fold, p=0.049) and beta-hemoglobin gene expression (betaeHb: 1.65+/-0.13 fold, p=0.026) and increased stat5 phosphorylation. These responses were also ameliorated by stat5.1 morpholino co-injection or treatment with a specific JAK2 inhibitor TG101209. jak2a(V581F) mRNA significantly reduced erythropoietin gene [0.24+/-0.03 fold (p=0.006)] and protein expression (control: 0.633+/-0.11; jak2a(V581F) mRNA: 0.222+/-0.07 mIU/mL, p=0.019). CONCLUSION: The zebrafish jak2a(V581F) model shared many features with human PV and might provide us with mechanistic insights of this disease.