pERK1/2 and Notch signaling work independently in Gpr171-mediated embryonic HSC specification. (A) Whole-embryo qPCR analysis of Notch signaling gene expression in WT embryos exposed to SCH772984 or vehicle control at 30 h postfertilization (hpf) (vehicle control, n = 3; SCH772984 treated, n = 3; two-tailed, unpaired Student’s t test, ns, not significant. Data are presented as means ± SD). (B) Confocal images of the DA in Tg(tp1:EGFP) embryos exposed to SCH772984 or vehicle control at 30 hpf (white arrowheads). (Scale bar, 100 μm.) (C) Qualification of EGFP+ cells in the DA from images in (B) (vehicle control, n = 14; SCH772984 treated, n = 10; two-tailed, ns, not significant. Data are presented as box plot). (D) Whole-embryo qPCR analysis of Notch signaling gene expression in gpr171−/− embryos at 30 hpf following injection of caMEK1 mRNA (gpr171−/−, n = 3; gpr171−/− + caMEK1 mRNA, n = 3; two-tailed, unpaired Student’s t test, ns, not significant. Data are presented as means ± SD). (E) Western blotting analysis for protein levels of pERK1/2, total ERK1/2, and Gapdh on WT embryos exposed to N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-Butyl Ester (DAPT) and vehicle controls at 30 hpf. (F) Qualification of pERK1/2 and ERK1/2 signal density from (E) relative to Gapdh (n = 3; two-tailed, unpaired Student’s t test, ns, not significant. Data are presented as means ± SD). (G) Western blotting analysis for protein levels of pERK1/2, total ERK1/2, and Gapdh in WT and gpr171−/− embryos in the Tg(hsp70:Gal4;UAS:NICD) background at 30 hpf after heat shock. (H) Qualification of pERK1/2 and ERK1/2 signal density from (G) relative to Gapdh (n = 3; One-Way ANOVA test, ns, not significant. Data are presented as means ± SD). (I) Western blotting analysis for protein levels of pERK1/2, total ERK1/2, and Gapdh at 30 hpf in WT, gpr171−/− embryos, and gpr171−/− embryos with her15.1 mRNA injection. (J) Qualification of pERK1/2 and ERK1/2 signal density from (I) relative to Gapdh (n = 3; One-Way ANOVA test, ***P < 0.001, ns, not significant. Data are presented as means ± SD).
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