A Dual Role of Evi-1 During Developmental Hematopoiesis

Abstract 765

The Evi-1 locus was originally identified as a common site of retroviral integration in murine myeloid tumors. Several reports associate Evi-1 expression with aggressiveness in myeloid leukemia. More recent studies suggest involvement of Evi-1 in developmental hematopoiesis. Here, we analyzed zebrafish embryo and human induced pluripotent stem (iPS) cells to understand how Evi-1 modulates early blood development. We found that the zebrafish homologue evi-1 co-localizes with the hematopoietic markers scl, gata1 and gata2 during embryogenesis, indicating a role in primitive hematopoiesis. To analyse the functional role of evi-1, we performed in vivo loss-of-function studies by injecting control and inhibitory morpholino oligonucleotides (MO) suppressing evi-1 pre-mRNA splicing in zebrafish zygotes. To control for off-target effects, three separate MO were designed and injected. N = 100 zebrafish embryos were analyzed per experiment in each group. In situ hybridization analyses performed in evi-1 morphants versus control fish revealed strongly impaired formation of myeloid embryonic cells as shown by reduced pu.1, mpo and l-plastin expression and suppressed numbers of DsRed positive lyz-expressing cells in Tg(lyz:DsRed) fish. Interestingly, scl expression was not affected, suggesting that evi-1 acts at later stages, regulating specification and/or survival/proliferation of pu.1 expressing progenitor cells. Consistent with this notion, no changes were observed in primitive erythroid progenitor cells as monitored by gata1 and hbae3 expression and flow cytometry analyses of GFP-positive globin-expressing cells in Tg(globin:eGFP) fish. Furthermore, runx1+/cmyb+ cells were strongly reduced, suggesting a separate role of evi-1 in definitive hematopoiesis. Supporting this hypothesis, evi-1 morphants displayed profound suppression of ikzf1+ lymphoid progenitors and rag1+ T-lymphocytes, which was not due to impaired thymus development, since foxn1 expression remained unaltered in evi-1 morphants. Previous reports in adult murine hematopoietic cells suggest that Evi-1 affects HSC proliferation through regulation of Gata2. Indeed, we could show that co-injection of gata2 mRNA was able to rescue the impaired myeloid as well as the HSC phenotype in evi-1 morphants. However, data generated by TUNEL assays indicate that evi-1 also confers apoptosis resistance to developing AGM-HSC. Whether this effect is also mediated by gata2 is currently being explored. We further investigated the effect of EVI-1 during differentiation of hematopoietic cells from human iPS cells. Confirming our results collected in zebrafish embryo, inhibition of EVI-1 strongly reduced the numbers of CD34+ and CD45+ blood progenitor cells and the generation of myeloid colonies in colony forming assays, while leaving SCL expression unaltered. Taken together, our data suggest that Evi-1 plays conserved roles in zebrafish and human hematopoietic development, regulating embryonic myelopoiesis and HSC formation while leaving primitive erythropoiesis unaltered. The precise molecular basis of evi-1 mediated effects on these separate phases of developmental hematopoiesis is subject of ongoing investigations.