Nlrc3 signaling is indispensable for hematopoietic stem cell emergence via Notch signaling in vertebrates

Abstract Hematopoietic stem and progenitor cells (HSPCs) generate all of the lineages of blood cells throughout the lifespan of vertebrates. The emergence of HSPCs occurs through the hemogenic endothelial (HE) to hematopoietic transition (EHT) process, which is finely-tuned by a variety of signaling pathways. Previous studies have emerged the essential roles of pattern-recognition receptors such as Toll-like receptors and RIG-I-like receptors in EHT. However, whether the nucleotide-binding domain leucine-rich repeat (NLR) containing family members participate in vertebrate embryonic hematopoiesis remains unclear. In this study, we determined that Nlrc3 was highly expressed in HSPCs differentiated from HE of vertebrates’ embryonic development in vivo and mouse embryonic stem cells and human induced pluripotent stem cells (iPSCs) in vitro. We then utilized morpholino and CRISPR-Cas9 technology to find that nlrc3 was required in definitive hematopoiesis and in primitive hematopoietic differentiation to neutrophils and macrophages in zebrafish embryos. Mechanistically, nlrc3 activated the Notch pathway and the downstream gene of Notch hey1 in the production of HSPCs. Furthermore, NF-kB signaling acted upstream of nlrc3 to enhance its transcriptional activity. Finally, we found that Nlrc3 signaling was conserved in the regulation of murine hematopoiesis. Taken together, our findings uncover an indispensable role of Nlrc3 signaling in HSPC emergence and provide new insights into inflammation-related hematopoietic ontogeny and the in vitro expansion of HSPCs.