Serca2 deletion in the mouse adult Bmi1+ compartment induces a lethal phenotype involving a severe gastric dysfunction

Figure 6 Impact of Serca2 conditional gene deletion in the lymphohematopoietic adult Bmi1⁺ cell lineages. A: Total cellularity in spleen (Sp), lymph nodes (LN) and thymus (Thy) of DSerca2^Bmi1homo [10 days post-tamoxifen (Tx) induction] (orange bars in all analyses) and control mice (blue bars in all analyses); B-D: Flow cytometry analysis of different thymic subpopulations including single-positive (SP) [cluster of differentiation 4 (CD4⁺) or 8 (CD8⁺)], double-positive (DP) (CD4⁺ CD8⁺) and double-negative (DN) (CD4^- CD8^-); B: Percental representation of SP, DP and DN populations; C: Percental representation of SP/DN ratios; D: Percental representation of DN subpopulations; E: Evaluation of T (CD4⁺ or CD8⁺) and B (B220⁺) cells in Sp and LN (n = 3 each; independent animals); F and G: Representative histograms (F) and mean fluorescence intensity (G) of CD5 expression in DP, SP and DN thymocyte subsets; notice the absence of CD5^high and reduced CD5^low cells in DSerca2^Bmi1homo Thy compared with controls. All comparisons were carried out in DSerca2^Bmi1homo (10 days post-Tx) compared with control mice (n = 3 each; independent animals); H: Proportions of total thymic T-cell receptor-αβ^high CD69⁺ positive selected cells; I: Gated dot plots for evaluating negatively selected thymocytes (Caspase3⁺ CD5^high CD69⁺) in both control and DSerca2^Bmi1homo thymuses; J: Thy histology of both control and DSerca2^Bmi1homo mice using an anti-pan cytokeratin (PanCK) marker for detecting cortical and anti-keratin 5 (K5) cytokeratin mAb for medullary epithelium; note the reduced cortical area and the enlarged medullary compartment in DSerca2^Bmi1 Thy compared with controls. Scale bar: 50 μm; K: Proportions of different thymic epithelial cells (TEC) subsets defined by the expression of major histocompatibility complex (MHC) II and ulex europaeus agglutinin (UEA)-1 cells markers in the total TEC (epithelial cell adhesion molecule⁺ CD45^-): Cortical TEC^low (UEA-1^- MHCII^low), medullary TEC^low (UEA-1⁺ MHCII^low), cortical TEC^high (UEA-1^- MHCII^high) and medullary TEC^high (UEA-1⁺ MHCII^high). Animals used for F-I and K were n = 3, controls; n = 2, DSerca2^Bmi1homo; age-matched animals; L: Numbers of Aire⁺ cells respect to K5⁺ area in thymic sections of DSerca2^Bmi1homo thymuses compared with controls ones (n = 2 each; age-matched animals). Data are expressed as mean ± SD. ^aP < 0.05. ^bP < 0.02. TEC: Thymic epithelial cell; Sp: Spleen; LN: Lymph nodes; Thy: Thymus; CD: Cluster of differentiation; DN: Double-negative; DP: Double-positive; TcR: T-cell receptor; K5: Keratin 5; PanCK: Pan cytokeratin; mTEC: Medullary thymic epithelial cell; cTEC: Cortical thymic epithelial cell.