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 ortholog of human JAK2^V617F (referred herewith jak2a^V581F) by site-directed mutagenesis and examined its relevance as a model of human PV. Materials and Methods: Zebrafish embryos at one-cell stage were injected with jak2a^V581F mRNA (200pg/embryo). 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 18-somites. Results: Injection with jak2a^V581F mRNA significantly increased erythropoiesis, as enumerated by flow cytometry based on gfp⁺ population in dissociated Tg(gata1:gfp) embryos. The response was reduced by stat5.1 morpholino coinjection (control: 4.37% ± 0.08%; jak2a^V581F injected: 5.71% ± 0.07%, coinjecting jak2a^V581F mRNA and stat5.1 morpholino: 4.66% ± 0.13%; p < 0.01). jak2a^V581F mRNA also upregulated gata1 (1.83 ± 0.08 fold; p = 0.005), embryonic α-hemoglobin (1.61 ± 0.12 fold; p = 0.049), and β-hemoglobin gene expression (1.65 ± 0.13-fold; p = 0.026) and increased stat5 phosphorylation. These responses were also ameliorated by stat5.1 morpholino coinjection 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.