Title: The zebrafish homologue of the murine EVI1 gene critically regulates hsc development
Abstract: EVI1 is one of the most potent oncogenes associated with myeloid malignancies and expressed in healthy adult hematopoietic stem cells (HSCs). Here, we employ the zebrafish model to explore the role of evi1 during blood development. Genetic modifications were obtained by morpholino oligonucleotide or mRNA injections or via heat-shock inducible transgenic lines. Hematopoiesis was analysed by in situ hybridisation, live cell imaging and flow cytometry on single-cell dissociated transgenic embryos. Sorted cells were further subjected to qRT-PCR analysis. Knockdown of evi1 strongly impaired embryonic myelopoiesis, while not altering primitive erythropoiesis. Furthermore, evi1 morphants showed reduced runx1/cmyb+ HSCs in the AGM, and later on showed less circulating globin or lyz cells and impaired megakaryopoiesis. Consistently, lack of ikaros+ lymphocyte precursors and rag+ T-lymphocytes was observed. Live imaging of double transgenic Tg(Kdrl:mKate-CAAX;cmyb:GFP) embryos indicated reduced emergence mKate+GFP+ putative HSCs from the ventral wall of the dorsal aorta (DA), suggesting defects in HSC budding from hemogenic endothelium (HE). Consistently, DA showed reduced expression of efnb2a and dlc. Furthermore, mKate+GFP+ cells sorted from evi1 morphants showed reduced expression of hematopoietic genes (runx1, cmyb) and enhanced expression of endothelial specific genes (kdrl, flt1, dab) as compared to corresponding control cells, suggesting that evi1 is required for full transition of HE cells to hematopoietic fate. On the molecular level, evi1 morphants showed reduced levels of notch pathway members and HSC formation was rescued by conditional heat-shock induction of NICD or its upstream regulator VEGF. Furthermore, the notch target gene gata2 also provided a partial HSC rescue. Taken together, our data suggest that evi1 regulates HSC specification from the HE via activation of NOTCH and downstream gata2. Currently, we analyze in more detail how evi1 regulates NOTCH on the molecular level and use the CRISPR/Cas9 system to generate evi1 mutant fish for further validation of our findings.