Redundant mechanisms driven independently by RUNX1 and GATA2 for hematopoietic development

RUNX1 is essential for the generation of hematopoietic stem cells (HSCs).Runx1null mouse embryos lack definitive hematopoiesis and die in mid-gestation. However, even though zebrafish embryos with arunx1W84X mutation have defects in early definitive hematopoiesis, somerunx1^W84X/W84Xembryos can develop to fertile adults with blood cells of multi-lineages, raising the possibility that HSCs can emerge without RUNX1. Here, using three new zebrafishrunx1^-/-lines we uncovered the compensatory mechanism forrunx1-independent hematopoiesis. We show that, in the absence of a functionalrunx1, acd41-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 thecd41-GFP⁺cells identified a set ofrunx1^-/--specific signature genes during hematopoiesis. Significantly,gata2b, which normally acts upstream ofrunx1for the generation of HSCs, was increased in thecd41-GFP⁺cells inrunx1^{- /-}embryos. Interestingly, genetic inactivation of bothgata2band its paralog,gata2a, did not affect hematopoiesis. However, knocking outrunx1and any three of the four alleles ofgata2aandgata2babolished definitive hematopoiesis.Gata2expression was also upregulated in hematopoietic cells inRunx1^-/-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.