GPR65 Suppresses Hematopoiesis in the Mouse and Zebrafish AGM.

(A) Representative flow cytometric plots of CD31⁺c-KIT⁺ and CD31⁺c-KIT⁺SCA1⁺ cell populations in control or Gpr65 shRNA-treated AGMs after 96 hr of culture.

(B) Quantitation of GFP⁺ cells with total live cells (9 litters: shLuc [n = 22 embryos]; shGpr65 [n = 26 embryos]).

(C) RT-PCR analysis of Gpr65 mRNA levels in FACS-sorted GFP⁺ cells (6 litters: shLuc [n = 15 embryos]; shGpr65 [n = 15 embryos]).

(D and E) Analysis of flow cytometry data expressed as percentage of CD31⁺c-KIT⁺ (D) and CD31⁺c-KIT⁺Scal1⁺ (E) cells in GFP⁺ cells (D: 9 litters: shLuc [n = 22 embryos]; shGpr65 [n = 26 embryos]; E: 7 litters: shLuc [n = 18]; shGpr65 [n = 22]).

(F) Representative images of ISH with the HSPC markers Runx1/cMyb at 36 hr post-fertilization.

(G) Quantitation of ISH data expressed as percentage of embryos with high, medium, and low Runx1/cMyb staining in total embryos (ATG MO 0 ng [124 embryos]; ATG MO 4 ng [75 embryos]; ATG MO 6 ng [66 embryos]; SP MO 0 ng [97 embryos]; SP MO 4 ng [49 embryos]; SP MO 6 ng [58 embryos]). Gpr65_ATG MO: morpholino targeting the translation start site of Gpr65; Gpr65_SP MO: morpholino blocking the splicing of Gpr65.

Error bars represent SEM. p < 0.05; ***p < 0.001 (two-tailed unpaired Student′s t test).

Related to Figure 5: GPR65 suppresses Gata2 expression in the zebrafish embryo. (A) Representative images of in situ hybridization for Gata2 at 36 hours post fertilization. The red rectangle illustrates the region enriched in hemogenic endothelium. (B) Quantitative analysis of in situ hybridization data expressed as percentage of embryos with high, medium, and low Gata2 staining in total embryos (ATG_MO 0ng [n=104 embryos]; ATG_MO 4ng [n=22 embryos]; ATG_MO 6ng [n=40 embryos]). Error bars represent SEM. *, P < 0.05 (two-tailed unpaired Student’s t-test).