NR4A1 and NR4A2 orphan nuclear receptors regulate endothelial-to-hematopoietic transition in mouse hematopoietic stem cell specification.

Hematopoietic stem cells (HSCs) sustain life-long hematopoiesis and emerge during mid-gestation from hemogenic endothelial progenitors via an endothelial-to-hematopoietic transition (EHT). The full scope of molecular mechanisms governing this process remains unclear. The NR4A subfamily of orphan nuclear receptors act as tumor suppressors in myeloid leukemogenesis and have never been implicated in HSC specification. Here, we report that Nr4a1 and Nr4a2 expression is upregulated in hemogenic endothelium during EHT. Progressive genetic ablation of Nr4a gene dosage results in a gradual decrease in numbers of nascent c-Kit+ hematopoietic progenitors in developing embryos, c-Kit+ cell cluster size in the dorsal aorta, and a block in HSC maturation, revealed by an accumulation of pro-HSCs and pre-HSC-type I cells and decreased numbers of pre-HSC-type II cells. Consistent with these observations, cells isolated from embryonic day 11.5 Nr4a1-/-; Nr4a2-/- aorta-gonads-mesonephros are devoid of in vivo long-term hematopoietic repopulating potential. Molecularly, employing spatial transcriptomic analysis we determined that the genetic ablation of Nr4a1 and Nr4a2 prevents Notch signaling from being downregulated in intra-aortic clusters and thus for pro-HSCs to mature into HSCs. Interestingly, this defect is partially rescued by ex vivo culture of dissected aorta-gonads-mesonephros with SCF, IL3 and FLT3L, which may bypass Notch-dependent regulation. Overall, our data reveal a role for the NR4A family of orphan nuclear receptors in EHT.