Haematopoietic stem cell (HSC) homeostasis is tightly controlled by growth factors, signalling molecules and transcription factors. Definitive HSCs derived during embryogenesis in the aorta-gonad-mesonephros region subsequently colonize fetal and adult haematopoietic organs. To identify new modulators of HSC formation and homeostasis, a panel of biologically active compounds was screened for effects on stem cell induction in the zebrafish aorta-gonad-mesonephros region. Here, we show that chemicals that enhance prostaglandin (PG) E2 synthesis increased HSC numbers, and those that block prostaglandin synthesis decreased stem cell numbers. The cyclooxygenases responsible for PGE2 synthesis were required for HSC formation. A stable derivative of PGE2 improved kidney marrow recovery following irradiation injury in the adult zebrafish. In murine embryonic stem cell differentiation assays, PGE2 caused amplification of multipotent progenitors. Furthermore, ex vivo exposure to stabilized PGE2 enhanced spleen colony forming units at day 12 post transplant and increased the frequency of long-term repopulating HSCs present in murine bone marrow after limiting dilution competitive transplantation. The conserved role for PGE2 in the regulation of vertebrate HSC homeostasis indicates that modulation of the prostaglandin pathway may facilitate expansion of HSC number for therapeutic purposes.

During vertebrate embryogenesis, hematopoietic stem cells (HSC) arise in the aorta-gonads-mesonephros (AGM) region. A zebrafish chemical genetic screen identified compounds that regulate blood flow as modulators of HSC formation. silent heart (sih) embryos that lack a heartbeat and blood circulation exhibited severely reduced HSCs. Blood flow modifiers exerted their effects after the onset of heartbeat; however, nitric oxide (NO) donors affected HSC induction even when treatment occurred prior to the initiation of circulation, and rescued HSCs in sih mutants. NO synthase (Nos) inhibitors and morpholino-knockdown of nos1 (nnos/enos) blocked HSC development. Embryonic transplantation assays demonstrated a cell-autonomous requirement for nos1. Nos3 (eNos) was expressed in HSCs in the murine AGM. Intrauterine Nos inhibition or Nos3 deficiency in mice resulted in the absence of hematopoietic clusters and reduced transplantable progenitors and HSCs. This work links blood flow to AGM hematopoiesis, and identifies NO as a conserved downstream regulator of HSC development.