pcsk1nl cooperates with Gpr171 to coordinate HSC specification. (A) WISH for cmyb in the VDA at 30 h postfertilization (hpf) following injection of pcsk1nl morpholino (MO) and standard control MO (black arrowheads), and its qualitative phenotypic distribution plot of embryos scored with low, medium, or high cmyb expression (control MO, n = 63; pcsk1nl MO, n = 65). (Scale bar, 200 μm.) (B) WISH for cmyb in the CHT at 72 hpf following injection of pcsk1nl and standard control MOs (black arrowheads), and its qualitative phenotypic distribution plot of embryos scored with low, medium, or high cmyb expression at 72 hpf (control MO, n = 44; pcsk1nl MO, n = 36). (Scale bar, 200 μm.) (C) Confocal images of HSCs in the VDA at 30 hpf following injection of pcsk1nl and standard control MOs into Tg(runx1:EGFP;kdrl:mCherry) (white arrowheads). Runx1+ cells are in green, and Kdrl+ cells are in red. (Scale bar, 50 μm.) (D) Qualification of Runx1+Kdrl+ cells from images in (C) (control MO, n = 8; pcsk1nl MO, n = 8; two-tailed, unpaired Student’s t test, ***P < 0.001. Data are presented as box plot). (E) WISH for cmyb in the VDA at 30 hpf following injection of pcsk1nl 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 = 44; WT + pcsk1nl mRNA, n = 51; gpr171−/−, n = 60; gpr171−/− + pcsk1nl mRNA, n = 48). (Scale bar, 200 μm.) (F) Confocal images of HSCs in the VDA at 30 hpf following injection of pcsk1nl 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. (Scale bar, 50 μm.) (G) Qualification of Runx1+Kdrl+ cells from images in (F) (WT, n = 10; WT + gpr171 mRNA, n = 7; gpr171−/−, n = 10; gpr171−/− + gpr171 mRNA, n = 7; One-Way ANOVA test, **P < 0.01, ns, not significant. Data are presented as box plot). (H) WISH for cmyb in the VDA at 30 hpf following coinjection of pcsk1nl MO with or without pcsk1nl mRNA and control MO (black arrowheads), and its qualitative phenotypic distribution plot of embryos scored with low, medium, or high cmyb expression (control MO, n = 78; pcsk1nl MO, n = 58; pcsk1nl MO + pcsk1nl mRNA, n = 59). (Scalebar, 200 μm.) (I) Confocal images of HSCs in the VDA at 30 hpf following coinjection of pcsk1nl MO with or without pcsk1nl mRNA, and control MO into the Tg(runx1:EGFP;kdrl:mCherry) embryos (white arrowheads). Runx1+ cells are in green, and Kdrl+ cells are in red. (Scale bar, 50 μm.) (J) Qualification of Runx1+Kdrl+ cells from images in (I) (control MO, n = 10; pcsk1nl MO, n = 12; pcsk1nl MO + pcsk1nl mRNA, n = 12; One-Way ANOVA test, *P < 0.05, **P < 0.01, ns, not significant. Data are presented as box plot).
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