Fig. 7

STAT3 Is Activated upon ifng1-2 Induction and Positively Regulates HSC Development

(A and B) ifng1-2 overexpression stimulates Stat3 activation in the AGM region. (A) Immunofluorescent staining of phosphorylated Stat3 (pStat3, green) in embryos without (A) and with (B) the hsp:ifng1-2 transgene. All embryos were heat shocked at 24 and 48 hpf and harvested at 52–54 hpf. The AGM region is recognizable by the vascular expression of kdrl:HRAS-mCherry (red).

(C–E) Ifng1-2 and Notch signaling regulate Stat3 activation. (C) Western blot analysis of pStat3, total Stat3, and actin expression in the dissected yolk-spanning region of the trunk. Dissected area is outlined by dashed lines in the embryo illustration; 36 hpf embryos were heat shocked for 5 and 8 hr for ifng1-2 and NICD overexpression, respectively. DMSO and DAPT treatments were done from 24–48 hpf. (D) Relative expression of pStat3 normalized to Actin. (E) Relative expression of total Stat3 normalized to Actin. Values represent means ± SEM, n = 3–4 independent experiments, n.s. p > 0.05, p d 0.05, p d 0.01.

(F–I) Stat3 inhibition causes a reduction in HSC numbers that cannot be restored by ifng1-2 overexpression. itga2b:EGFP⁺ (green) kdrl:HRAS-mCherry⁺ (red) HSCs (white arrowheads) in control embryos (not carrying the hsp:ifng1-2-V5 transgene) treated with DMSO (F) or Stat3 inhibitor (inh) (G) and Tg(hsp:ifng1-2-V5) embryos treated with Stat3 inh (H). All embryos were heat shocked at 24 and 48 hpf and imaged at 52–54 hpf. DMSO and Stat3 inh treatments started at 24 hpf. (I) Numbers of itga2b:EGFP⁺kdrl:HRAS-mCherry⁺ cells per 500 µm aortic length are shown as means ± SEM, n = 19–24 embryos, n.s. p > 0.05, p d 0.01.