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Fig. 3 Gpr171 regulates embryonic HSC development via ERK1/2 signaling activation. (A) Western blotting analysis for protein levels of pERK1/2, total ERK1/2, and Gapdh in WT and gpr171−/− embryos following injection of the constitutively active form of MEK1 (caMEK1) mRNA and their siblings at 30 h postfertilization (hpf). (B) Qualification of signal density of pERK1/2 and ERK1/2 from (A) relative to Gapdh (n = 4; One-Way ANOVA test, *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant. Data are presented as means ± SD). (C) WISH for cmyb in the VDA at 30 hpf following injection of caMEK1 mRNA into WT and gpr171−/− embryos (black arrowheads), and its qualitative phenotypic distribution plot of embryos scored with low, medium, or high cmyb expression (WT, n = 112; WT + caMEK1 mRNA, n = 68; gpr171−/−, n = 112; gpr171−/− + caMEK1 mRNA, n = 80). (Scale bar, 200 μm.) (D) Confocal images of HSCs in the VDA at 30 hpf following injection of caMEK1 mRNA into WT and gpr171−/− embryos in Tg(runx1:EGFP;kdrl:mCherry) background (white arrowheads). Runx1+ cells are in green, and Kdrl+ cells are in red. (Scalebar, 50 μm.) (E) Qualification of Runx1+Kdrl+ cells from images in (D) (WT, n = 12; WT + caMEK1 mRNA, n = 17; gpr171−/−, n = 10; gpr171−/− + caMEK1 mRNA, n = 8; One-Way ANOVA test, *P < 0.05, ***P < 0.001. Data are presented as box plot).