Distinct roles of Atf3, Zfp711 and Bcl6b in early embryonic hematopoietic and endothelial lineage specification

Hematopoiesis occurs in three consecutive overlapping waves in mammals, regulated by transcription factors. We investigated the role of three relatively poorly studied transcription factors in early embryonic hematopoietic development at single-cell resolution: Atf3, Zfp711 and Bcl6b. These transcription factors are upregulated early in development, when hematopoietic and endothelial lineages separate from cardiac and other mesodermal lineages. We combined multiplexed single-cell RNA sequencing and flow cytometric analysis with knockouts in in vitro differentiating mouse embryonic stem cells to dissect the function of these transcription factors in lineage specification. ΔAtf3 cells showed increased mesodermal differentiation but decreased endothelial cells and erythro-myeloid progenitors, accompanied by aberrant interferon signaling. Mechanistically, loss of Atf3 disrupted key hematopoietic regulatory genes (Runx1, Egr1, Jun, Fos, Mafb and Batf3) required for the formation of erythro-myeloid progenitors. ΔZfp711 cells exhibited increased blood progenitors and erythroid cells, but decreased endothelial cells, with a striking shift from Hoxa+ mesoderm (allantois and limb mesoderm) to Hoxb+ mesoderm (mesenchyme and epicardium). Notably, Zfp711 binds the Atf3 promoter, suggesting a hierarchical regulation. In contrast, ΔBcl6b had no observable effects on early hematopoiesis, despite specific expression in hemato-endothelial progenitors.