Zebrafish gpr182 mutant embryos exhibit increased HE and HSC formation. (A) Brightfield images of WISH for cmyb expression in wild-type and gpr182–/– embryos at 36 hpf. White arrows point to cmyb positive cells in the trunk. N/N, number of embryos showing representative phenotype/total number of embryos examined. Two independent experiments were performed with similar results. (B) qPCR analysis of cmyb mRNA levels from wild-type and gpr182–/– embryos at 36 hpf. N = 5 biological replicates. A delta delta Ct (ΔΔCt) analysis was performed and wild-type expression levels were set at 1. Data are mean ± s.d., and a two-tailed Student’s t test was used to calculate P values. (C) Confocal images of Tg(cd41:EGFP); Tg(kdrl:Hsa.HRASmCherry) wild-type and gpr182–/– embryos in the trunk at 48–52 hpf. White arrows point to cd41/kdrl double-positive cells in the trunk. (D) Number of cd4l/kdrl double-positive cells in the trunk (six somites). Wild-type N = 10, gpr182–/–N = 13. N obtained from three independent clutches. (E) Brightfield images of WISH for cmyb expression in wild-type and gpr182–/– larvae at 96 hpf. N/N, number of embryos showing representative phenotype/total number of embryos examined. Two independent experiments were performed with similar results. (F) Confocal images of Tg(cd41: EGFP); Tg(kdrl: Hsa.HRASmCherry) wild-type and gpr182–/– larvae in the tail at 72 hpf. White arrows point to weak EGFP positive HSCs in the tail. (G) Number of weak cd41:EGFP positive HSCs in the tail (4 somites) of wild-type and gpr182–/– embryos. Wild-type (N = 12) and gpr182–/– (N = 17), from three independent clutches. (H) Time-lapse confocal images of Tg(cmyb:GFP); Tg(kdrl:Hsa.HRASmCherry) wild-type and gpr182–/–embryos at 36 hpf in the trunk. White arrows point to cmyb/kdrl double-positive HE/HSCs. The red, orange, green, blue and purple boxes in the above panels are enlarged in the bottom panels, respectively. Yellow (1′–2′′) and white (3′–5′′) arrowheads in the bottom panels point to HE/HSCs of the wild-type and gpr182–/–embryo, respectively. (I) Confocal images of Tg(cmyb:GFP); Tg(kdrl:Hsa.HRASmCherry) uninjected and gpr182 mRNA injected embryos at 36 hpf. White arrows point to cmyb/kdrl double positive cells in the trunk. (J) Quantification of cmyb/kdrl double-positive cells in the trunk (six somites). Uninjected embryos (N = 7) and gpr182 wild-type mRNA injected embryos (N = 8), from three independent clutches. Data are mean ± s.d.. A two-tailed Student’s t test was used to calculate p-values. Scale bars, 50 μm. Anterior to the left, dorsal to the top. DA, dorsal aorta; CV, caudal vein; PCV, posterior cardinal vein; VDA, ventral wall of DA.
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