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

<b>Objective :</b> The Janus kinase 2 (JAK2) is important for embryonic primitive hematopoiesis. A gain-of-function JAK2 (JAK2<i>^V617F</i>) mutation in human is pathogenetically linked to polycythemia vera (PV). In this study, we generated a zebrafish ortholog of human JAK2^<i>V617F</i> (referred herewith jak2a^<i>V581F</i>) by site-directed mutagenesis and examined its relevance as a model of human PV.<br /><br /><b>Materials and Methods :</b> Zebrafish embryos at one-cell stage were injected with jak2a^<i>V581F</i> 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.<br /><br /><b>Results :</b> Injection with jak2a^<i>V581F</i> mRNA significantly increased erythropoiesis, as enumerated by flow cytometry based on gfp+ population in dissociated Tg(<i>gata1:gfp</i>) embryos. The response was reduced by <i>stat5.1</i> morpholino coinjection (control: 4.37% ± 0.08%;<i> jak2a</i>^<i>V581F</i> injected: 5.71% ± 0.07%, coinjecting <i>jak2a</i>^<i>V581F</i> mRNA and <i>stat5.1</i> morpholino: 4.66% ± 0.13%; <i>p</i> < 0.01). <i>jak2a</i>^<i>V581F</i> mRNA also upregulated <i>gata1</i> (1.83 ± 0.08 fold; <i>p</i> = 0.005), <i>embryonic α-hemoglobin</i> (1.61 ± 0.12 fold; <i>p</i> = 0.049), and <i>β-hemoglobin</i> gene expression (1.65 ± 0.13–fold; <i>p</i> = 0.026) and increased stat5 phosphorylation. These responses were also ameliorated by <i>stat5.1</i> morpholino coinjection or treatment with a specific JAK2 inhibitor, TG101209. <i>jak2a</i>^<i>V581F</i> mRNA significantly reduced erythropoietin gene (0.24 ± 0.03 fold; <i>p</i> = 0.006) and protein expression (control: 0.633 ± 0.11;<i> jak2a</i>^<i>V581F</i> mRNA: 0.222 ± 0.07 mIU/mL; <i>p</i> = 0.019).<br /><br /><b>Conclusion : </b>The zebrafish <i>jak2a</i>^<i>V581F</i> model shared many features with human PV and might provide us with mechanistic insights of this disease.<br />