Adenosine-regulated HSPC emergence is a cAMP–PKA-dependent pathway. (A) cAMP luminescence assay in HAECs (Student’s t test: *, P < 0.05; n = 3). (B) H89 inhibited CXCL8 level induced by BAY 60-6583 in HAECs (Student’s t test: *, P < 0.05; **, P < 0.01; n = 3). (C–F) Expression of runx1/cmyb at 36 hpf. Wild-type embryos were treated with DMSO (C), 20 µM NECA (D), or 1 µM H89 (E) or co-treated with NECA and H89 (F) from 5-somite to 36 hpf. (G–J) Expression of runx1/cmyb at 36 hpf. Uninjected wild-type embryos treated with DMSO (G) or 1 µM forskolin (H) and embryos injected with A2b MO treated with DMSO (I) and forskolin (J) are shown. (K–L′) Confocal imaging of Tg(sclβ:d2eGFP; flk1:mcherry) embryos at 30 hpf. Embryos treated with DMSO (K and K′) or 1 µM H89 (L and L′) are shown. Arrowheads indicate the hemogenic endothelial cells marked by sclβ:GFP+. Dashed lines mark the somite boundaries. (M) Summary of the number of sclβ:GFP+ hemogenic endothelial cells per somite (Student’s t test: *, P < 0.05; n = 5 per group). The results are presented as mean ± SE. Bars, 100 µm.
