NO signaling is directly regulated by klf2a in the control of artery maturation and maintenance of HSC programming in zebrafish. (A-C) DAF-FM imaging showed reduced NO production in tnnt2a- and klf2aMO-injected embryos at 36 hpf. Yellow arrows mark normal NO production in the notochord area; red arrows mark reduced NO production. (D) Quantitative RT-PCR analysis of nos1 and nos2 expression in the dissected trunk region of control and klf2aMO embryos at 36 hpf (mean ± SD, t test, *P < .05, **P < .01, n = 3). (E) ChIP assay showing that Klf2a directly binds to nos1 and nos2b promoter region. (F-K) Rescue of runx1 expression in tnnt2a- and klf2aMO-injected embryos by klf2a mRNA or SNAP treatment. (F,G,I) Expression of runx1 in HSCs of tnnt2aMO-injected embryos (G) was partially rescued by coinjection of klf2a mRNA (I) at 36 hpf. (J-K) SNAP treatment at tailbud stage can rescue runx1 expression in tnnt2aMO (G,J) and klf2aMO (H,K) injected embryos. (L) Model of blood flow-induced klf2a-NO signaling in HSC maintenance.