MKs express S1pr1, and S1pr1-deficient mice display severe thrombocytopenia. (A) Relative expression of S1P receptor mRNA by FL-derived mature and immature MKs. (B) Relative expression of S1P receptor mRNA in human megakaryocytic cell lines. (A and B) Data are representative of three independent experiments with triplication. (C) Representative immunostaining of S1pr1 in immature and mature FL-derived MKs. WT MKs stained with irrelevant control IgG or anti-S1pr1–stained S1pr1-null MKs served as controls. (D and E) Expression of S1pr1 in murine BM (D) and S1PR1 in human BM sections (E). Arrowheads indicate MKs. CD42c is the marker for MKs. All MKs examined stained positive for S1pr1 or S1PR1. Bars, 10 µm. (F) Platelet counts in peripheral blood. n = 15 for WT BM chimaeras; n = 5 for S1pr2^−/− BM chimaeras; n = 3 for S1pr4^−/− BM chimaeras. (G) Platelet counts in peripheral blood. n = 8 for WT; n = 6 for S1pr1^+/−; n = 15 for WT BM chimaeras; n = 9 for S1pr1^+/− BM chimaeras; n = 14 for S1pr1^−/− BM chimaeras. (H) Platelet counts in chimaeras after transferring S1pr1^fl/fl BM cells transduced with lenti-GPIIb-cre or empty control vectors into irradiated recipient mice (left). Expression of S1pr1 in platelets in chimaeras after transferring S1pr1^fl/fl BM cells transduced with lenti-GPIIb-cre or empty control vectors. β-Actin served as loading control (right). n = 3 per genotype. (I) Percentage of EGFP⁻ platelets in chimaeras after hematopoietic reconstitution of lethally irradiated mice with EGFP⁺ S1pr1^+/+ BM cells and EGFP⁻ S1pr1^−/− FL cells at a ratio of 20:1. The EGFP⁻ S1pr1^−/− FL cells were transduced with lenti-GpIbα-S1pr1 or empty control vectors before transplantation. n = 3 per genotype. All error bars indicate SEM.