Fig. 4

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).