Ninjurins regulate hematopoietic stem cell amplification through canonical WNT signaling independent of plasma membrane rupture

Ninjurin-1 (NINJ1), a protein required for plasma membrane rupture (PMR) during inflammasome-mediated cell death, has no known role in hematopoiesis. Here, using zebrafish and human hematopoietic stem/progenitor cells (HSPCs), we identify an evolutionarily conserved, PMR-independent function of Ninjurins in HSPC development. Ninj1 deficiency reduced HSPC, neutrophil and erythrocyte numbers, whereas PMR-deficient Ninj1 mutants demonstrated that this function is independent of PMR. Ninj1 deficiency also impaired immune cell recruitment and increased susceptibility to bacterial infection. Mechanistically, Ninj1 and Ninj2 cooperatively promoted early HSPC amplification through canonical WNT signaling. Pharmacological and genetic activation of WNT signaling rescued hematopoietic defects in Ninjurin-deficient larvae, whereas inhibition of WNT abolished the effects of Ninj1 gain of function. Human CD34⁺ HSPCs showed reduced expansion and impaired WNT signaling following NINJ1/2 deficiency, demonstrating conservation of this pathway. These findings identify Ninjurins as regulators of HSPC expansion with potential applications in regenerative medicine. Here they show that Ninjurins promote the expansion of blood-forming stem cells by activating WNT signaling independently of their role in cell death. This evolutionarily conserved mechanism identifies Ninjurins as potential targets for regenerative medicine and hematopoietic disorders.