1 I). These findings demonstrate that S1pr1 expressed by the MK lineage intrinsically controls platelet homeostasis. Normal MK development, platelet life span, and serum TPO levels in S1pr1-deficient mice What could be the reason for the severe thrombocytopenia in the absence of S1pr1? First we selleck chemicals Vorinostat showed that the life spans of platelets from S1pr1+/+, S1pr1+/?, or S1pr1?/? chimaeras and between WT and S1pr1+/? mutant mice was similar, excluding a reduced life span as cause for the reduced platelet counts (Fig. 2 A). We also excluded a defect in the release of TPO, the principle regulator of thrombopoiesis (Kaushansky, 2005a), as cause for the thrombocytopenia in S1pr1-null mutants (Fig. 2 B). Finally, we also could not find evidence for a gross defect in MK development, as we found similar numbers of megakaryocytic progenitor cells in WT and S1pr1?/? FL cells populations (Fig.

2 C) and a normal differentiation of WT and S1pr1?/? precursor cells into MKs both in vitro (Fig. 2 D) and in vivo (Fig. 2, E and F). Figure 2. Loss of S1pr1 does not change platelet life span, serum TPO levels, and MK development. (A) Platelet life span assays in the indicated genotypes. n = 3�C5 per genotype. (B) Serum TPO levels. n = 3�C5 per genotype. (C) Quantification of CFU-MK … Loss of S1pr1 increases MK size but has no effect on positioning and motility of MKs in vivo Next we examined whether S1pr1 controls platelet biogenesis for example by modulating MK motility or their positioning within the BM compartment. To address this question, we performed MP-IVM of calvarian BM (Junt et al.

, 2007) of two different sets of S1pr1+/+ or S1pr1?/? chimaeras, in which MKs and their progeny were genetically marked: (a) S1pr1+/+ or S1pr1?/? CD41-YFPki/+ FL chimaeras, in which MKs and platelets express the YFP driven from the endogenous CD41 gene locus (Zhang et al., 2007) and (b) S1pr1+/+ or S1pr1?/? lenti-GpIb���Cenhanced GFP (EGFP) BM chimaeras, in which MKs and platelets express EGFP under the transcriptional control of the murine GpIb�� promoter (Lavenu-Bombled et al., 2007). The experiments revealed neither differences in MK size nor in their positioning or motility when we compared S1pr1+/+ CD41-YFPki/+ or S1pr1+/+ lenti-GpIb��-EGFP chimaeras and naive (nontransplanted) S1pr1+/+ CD41-YFPki/+ or platelet factor 4 (Pf4)�CEYFP transgenic mice, in which EYFP is driven by the MK-specific Pf4 promoter, which allowed excluding a major influence of irradiation and BM transplantation (Fig.

3, A�CD). As reported previously (Junt et al., 2007), S1pr1+/+ MKs were large, mostly Cilengitide sessile cells always located in close proximity to BM sinusoids (Fig. 3, A and D�CG). In S1pr1?/? chimaeras and S1pr1+/? mice, MKs were significantly larger compared with S1pr1+/+ chimaeras, whereas the position and motility of MKs was similar among all genotypes (Fig. 3, E�CG).