Abstract RUNX1 is essential for the generation of hematopoietic stem cells (HSCs). Runx1 null mouse embryos lack definitive hematopoiesis and die in mid-gestation. However, even though zebrafish embryos with a runx1 W84X mutation have defects in early definitive hematopoiesis, some runx1 W84X/W84X embryos can develop to fertile adults with blood cells of multi-lineages, raising the possibility that HSCs can emerge without RUNX1. Here, using three new zebrafish runx1 -/- lines we uncovered the compensatory mechanism for runx1 -independent hematopoiesis. We show that, in the absence of a functional runx1 , a cd41 -GFP + population of hematopoietic precursors still emerge from the hemogenic endothelium and can colonize the hematopoietic tissues of the mutant embryos. Single-cell RNA sequencing of the cd41 -GFP + cells identified a set of runx1 -/- -specific signature genes during hematopoiesis. Significantly, gata2b , which normally acts upstream of runx1 for the generation of HSCs, was increased in the cd41 -GFP + cells in runx1 - /- embryos. Interestingly, genetic inactivation of both gata2b and its paralog, gata2a , did not affect hematopoiesis. However, knocking out runx1 and any three of the four alleles of gata2a and gata2b abolished definitive hematopoiesis. Gata2 expression was also upregulated in hematopoietic cells in Runx1 -/- mice, suggesting the compensatory mechanism is conserved. Our findings indicate that RUNX1 and GATA2 serve redundant roles for HSC production, acting as each other’s safeguard. Key points Existence of RUNX1-independent mechanisms for the generation of HSCs and the development of functional definitive hematopoietic cells GATA2 and RUNX1 functionally complement each other for their respective roles during hematopoiesis

Redundant mechanisms driven independently by RUNX1 and GATA2 for hematopoietic development