Rescue of HSCs in spt5^m806 by modulating the activity of Stat3 and TGFβ signaling. (A, top) Partial sequence alignment of mouse and zebrafish STAT3. Two residues (red) are mutated in Stat3-CA (blue). (Bottom) WISH for runx1 in 36-hfp uninjected embryos, embryos injected with mRNA encoding Stat3-CA or wild-type STAT3 (STAT-WT). (B) WISH for runx1 in 36-hpf embryos treated with DMSO, individual TGFβ inhibitor (LY2157299 and SB505124), or Stat3-CA injection together with a TGFβ inhibitor (SB505124 + Stat3-CA). (C) Quantification of rescue efficiency by the percentage of spt5^m806 embryos exhibiting increased runx1 staining under different treatment conditions (10-15 embryos per group, n = 3, mean ± SEM, **P < .01). (D) WISH for runx1 in untreated (no HS) vs HS-treated embryos carrying HS-inducible constitutive Tgfbr1 (hsp70:caAlk5). HS treatment was performed by incubating transgenic embryos at 37°C for 1 hour at 14 hpf. (E) Fluorescent microscopy imaging of Tg(kdrl:dsRed; cd41:GFP) embryos at 36 hpf under different treatment conditions. Arrowheads indicate nascent HSCs that are double positive for dsRed and GFP. Scale bar, 50 μM. (F) Double immunofluorescent staining for GFP (green) and pH3 (red) in CHT in 48 hpf Tg(cd41:GFP) embryos. Arrowheads indicate cells that are GFP⁺pH3⁺. Scale bar, 50 μM. (G-H) Quantification of the results from panels E and F, respectively (10-15 embryos per group, n = 3, mean ± SEM, **P < .01).