Gpr171 is essential for embryonic HSC development in zebrafish. (A) Schematic representation of the generation of gpr171 mutant zebrafish line. Illustration was created with BioRender.com. (B) Whole-mount in situ hybridization (WISH) for cmyb in the ventral wall of the dorsal aorta (VDA) of WT and gpr171−/− embryos at 30 h postfertilization (hpf) (black arrowheads), and its qualitative phenotypic distribution plot of embryos scored with low, medium, or high cmyb expression (WT, n = 47; gpr171−/−, n = 68). (Scale bar, 200 μm.) (C) WISH for cmyb in the caudal hematopoietic tissue (CHT) of WT and gpr171−/− embryos at 72 hpf (black arrowheads), and its qualitative phenotypic distribution plot of embryos scored with low, medium, or high cmyb expression (WT, n = 113; gpr171−/−, n = 85). (Scale bar, 200 μm.) (D) Confocal images of hematopoietic stem cells (HSCs) in the VDA of WT and gpr171−/− embryos in Tg(runx1:EGFP;kdrl:mCherry) background at 30 hpf (white arrowheads). Runx1+ cells are in green, and Kdrl+ cells are in red. (Scale bar, 50 μm.) (E) Qualification of Runx1+Kdrl+ cells from images in (D) (WT, n = 10; gpr171−/−, n = 9; two-tailed, unpaired Student’s t test, ***P < 0.001. Data are presented as box plot). (F) WISH for cmyb in the VDA at 30 hpf following injection of gpr171 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 = 43; WT + gpr171 mRNA, n = 66; gpr171−/−, n = 52; gpr171−/− + gpr171 mRNA, n = 52). (Scale bar, 200 μm.) (G) WISH for cmyb in the CHT at 72 hpf following injection of gpr171 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 = 36; WT+ gpr171 mRNA, n = 28; gpr171−/−, n = 38; gpr171−/− + gpr171 mRNA, n = 43). (Scale bar, 200 μm.) (H) Confocal images of HSCs in the VDA at 30 hpf following injection of gpr171 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.) (I) Qualification of Runx1+Kdrl+ cells from images in (H) (WT, n = 11; WT+ gpr171 mRNA, n = 13; gpr171−/−, n = 10; gpr171−/− + gpr171 mRNA, n = 11; One-Way ANOVA test, **P < 0.01, ***P < 0.001, ns, not significant. Data are presented as box plot). (J) WISH for cmyb in the VDA at 30 hpf following injection of gpr171 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 = 50; gpr171 MO, n = 57). (Scale bar, 200 μm.) (K) Confocal images of HSCs in the VDA at 30 hpf following injection of gpr171 and standard control MOs into Tg(runx1:EGFP;kdrl:mCherry) embryos at 30 hpf (white arrowheads). Runx1+ cells are in green, and Kdrl+ cells are in red. (Scale bar, 50 μm.) (L) Qualification of Runx1+Kdrl+ cells from images in (K) (control MO, n = 14; gpr171 MO, n = 15; two-tailed, unpaired Student’s t test, ***P < 0.001. Data are presented as box plot).
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