Differential Role of the Transcription Factor ZBP-89 in Hemangioblast Fate Determination: ZBP-89 Is a Direct Regulator of SCL.

Several molecular pathways have been identified that regulate distinct stages in the developmental progression from mesoderm to the formation of the hematopoietic and vascular lineages. Our previous work indicated that ectopic expression of the zinc finger transcription factor ZBP-89 promotes hematopoietic lineage development and represses endothelial cell lineage differentiation from hemangioblasts in murine embryonic stem cells. Here we evaluated the functional consequences of stable knockdown of ZBP-89 in embryonic stem cells (ESC) on hematopoietic and vascular development. Stable knock down of ZBP-89 in ESC significantly decreased the number of Blast Colony Forming Cells (BL-CFC) hemangioblasts, as well as primitive and definitive hematopoietic progenitor colonies BFU-E, GM-CFU, G-CFU, M-CFU and GEMM-CFU in vitro. In contrast, sprouting angiogenesis was markedly increased in EB cultures. Flow cytometric analysis of the lineages derived from ZBP-89 deficient EB cultures showed that the early (C-kit+Sca-1+) and definitive (CD45+) hematopoietic stem cells populations were reduced, but the endothelial cell population (CD31+ VE-Cadherin+) was increased. RT-PCR analysis of EB cultures revealed a direct correlation between the expression levels of ZBP-89 and hematopoietic markers (including SCL and Runx1) but an inverse correlation with the vascular marker CD31, with no change in Oct4 expression level. To investigate the mechanism underlying the role of ZBP-89 in hematopoiesis, the effect of ZBP-89 on expression of SCL, a master regulator of hematopoiesis, was examined. The murine SCL promoter transduced into the ZBP-89-expressing MEL cell line drove luciferase gene expression. ZBP-89 knockdown in MEL cells markedly reduced SCL expression. ChIP analysis showed that endogenous ZBP-89 protein bound directly to the murine SCL promoter in MEL cells. Thus ZBP-89 plays a central role in fate determination of hemangioblasts; its induction suppresses angiogenesis but enhances differentiation of hemangioblasts along the hematopoietic pathway, an effect mediated through the regulated expression of SCL.