S1P₁is not expressed in murine MKs. (A) Expression of S1P₁ (red) and the MK marker CD41 (green) in spleen of mice with or without MK-selective (S1pr1^f/f:Pf4Cre⁺) or pan-hematopoietic (S1pr1^f/f:Mx1Cre⁺) S1P₁ deletion. Note S1P₁ expression in blood vessels and white pulp, but not in MKs, irrespective of gene deletion. Scale bars represent 50 μm. (B) Constitutive (left) and fingolimod-induced (1 mg/kg, 24 hours; right) S1P₁ signaling in spleens of S1P₁ signaling reporter mice.³⁴  Note constitutive and fingolimod-enhanced S1P₁ signaling (reflected by nuclear GFP accumulation in green) in blood vessels (in red) and cells within the white pulp, but not MKs (in blue). Scale bars represent 50 μm. (C) Abundance of S1PR transcripts in BM-derived MKs from S1pr1-deficient mouse lines relative to Gapdh. Note lack of S1pr1 expression (mRNA) or compensatory upregulation of other receptors after 3 days of culture. (D) Abundance of nonexcised S1pr1 in genomic DNA (gDNA) from BM-derived MKs from S1pr1-deficient mouse lines after 5 days of culture relative to pooled S1pr1^f/f littermate controls. (E) Relative abundance of nonexcised S1pr1 in genomic DNA from freshly isolated BM cells from S1pr1-deficient mice passed through 70-μm filters. Statistical analysis by Mann-Whitney U test. n.d., not detectable.