Figure 3. Cxcr4 is required for development of small pre-B cells.

a, Genomic PCR of WT and floxed alleles for Cxcr4 deletion in tail DNA and flow sorted large pre-B cells from indicated mice with Gapdh as control (n=3). b, Flow cytometric analysis of different developmental stages of B lymphopoiesis in the BM of wild-type (WT), mb1-cre^+/−, Cxcr4^fl/fl and Cxcr4^fl/fl- mb1-cre^+/− littermate control mice (n=9). B cell progenitors are defined as B220⁺CD19⁺, pro-B cells as B220⁺CD19⁺CD43⁺IgM⁻, large and small pre-B as B220⁺CD19⁺CD43⁻IgM⁻FSC^hi and B220⁺CD19⁺CD43⁻IgM⁻FSC^lo respectively and immature B cells as B220⁺CD19⁺CD43⁻IgM⁺. FSC, forward scatter. c, Absolute number of cells per mouse at different stages of B cell development in BM of WT, mb1-cre^+/−, Cxcr4^fl/fl and Cxcr4^fl/fl-mb1-cre^+/− mice (n=9)(Imm B, Immature B cell; Mat B, mature B cells. *P<0.001 compared to all the controls (WT, mb1-cre^+/−, Cxcr4^fl/fl). Data presented as average ± SD. d,e, Distribution of B cell progenitors in BM of Cxcr4^fl/fl (CXCR4 sufficient; d) and Cxcr4^fl/fl-mb1-cre^+/− (CXCR4 deficient; e) mice by confocal microscopy of corresponding BM sections (8μm thick femur) stained with antibodies to IL-7 (red), CXCL12 (blue), B220 (yellow), IgM (green) and DAPI (gray) (n=4). f, Percentage of B cell progenitors (B220⁺) in proximity to IL-7^−/loCXCL12⁺ and IL-7^hiCXCL12^+/−stroma (n=4 independent image). Data are presented as mean±SD. Each dot represents average distance obtained from each image of indicated genotype. P values were calculated by unpaired t-test.