Multiple Roles for the Zebrafish Homologue of the Murine Evi1 Gene during Primitive Myelopoiesis and HSC Development

The Evi1 locus was originally identified as a common site of retroviral integration in murine myeloid tumors. Several reports associate Evi1 expression with aggressiveness in myeloid leukemia. Since developmental pathways often reactivate in cancer, we hypothesized that Evi1 also plays critical roles during developmental hematopoiesis. Here, we employ the zebrafish model to study how evi1 modulates early blood development. We find that indeed zebrafish evi1 co-localizes with the hematopoietic markers scl, gata1, pu.1 and gata2 in the posterior lateral mesoderm and the rostral blood islands, indicating involvement in primitive hematopoiesis. Knockdown of evi1 via three independent Morpholino Oligonucleotides impairs embryonic myelopoiesis as shown by reduced pu.1, mpo and l-plastin staining, while not affecting hemangioblast formation and primitive erythropoiesis. Additionally, we observe reduced levels of cd41 expression upon evi1 knockdown, indicating that megakaryopoiesis is also impaired.

Notably, at later time-points, evi1 is also expressed in the AGM region and evi1 morphants show strong reduction of runx1/c-myb expression in this region, demonstrating an additional role in hematopoietic stem cell (HSC) formation. Consistently, evi1 morphants lack ikaros+ lymphocyte precursor cells and rag1+ T-lymphocytes, and less circulating globin+, lyz+ and cd41+ cells are detected in transgenic fish analyzed by flow cytometry at 5 days post fertilization. To dissect the mechanisms by which evi1 regulates HSC formation, we furthermore analyzed genes specifically expressed in the dorsal aorta, where HSCs emerge from the hemogenic endothelium. We hypothesize that evi1 is indeed regulating HSC specification from hemogenic endothelial cells, since detailed analyses show defective aortic expression of efnb2a and dlc. How this effect is mediated is currently being investigated. Furthermore, TUNEL, anti-activated Caspase-3, anti-phospho Histone H3 and BrdU assays show both increased apoptosis and decreased proliferation in the AGM region of evi1 morphants as compared to control injected fish, suggesting important additional effects of evi1 on HSC biology beyond the specification step. Similar effects are seen in the rostral blood islands and are possibly responsible for the observed reduction of primitive myeloid progenitors.

We also examined potential downstream targets of evi1. Previous reports in adult murine hematopoietic cells suggest that Evi1 affects HSC proliferation through regulation of Gata2. Indeed, gata2 co-injection enhances hematopoietic cell viability also in zebrafish embryos, and co-injection of gata2 mRNA is able to fully rescue both primitive myelopoiesis and HSC formation. We are currently investigating whether gata2 expression can compensate evi1 requirement during HSC specification or, alternatively, might rescue HSC numbers by amplifying residual HSCs that escape evi1 inhibition.

Taken together, our data demonstrate that evi1 plays multiple roles during hematopoietic development, critically regulating the biology of primitive myeloid progenitors and pre-formed HSCs, as well as HSC specification from the hemogenic endothelium. Currently, we analyze the molecular mechanisms that mediate evi1 effects during these different regulatory steps and use the CRISPR/Cas9 system to generate evi1 mutant fish for further validation of our findings.