EphB receptors, mainly EphB3, contribute to the proper development of cortical thymic epithelial cells

Figures & data

FIGURE 1. Percentages and numbers of total TECs (EpCAM⁺CD45⁻ cells) in WT and EphB-deficient thymuses throughout fetal development. (A) Comparative analysis of the proportions of EpCAM+CD45− cells between WT and mutant fetal thymuses. (B) Comparative analysis of the numbers of total TECs between WT and mutant thymuses. The significance of the Student's t-test probability is indicated as *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.005; or #p ≤ 0.05; ##p ≤ 0.01; ###p ≤ 0.005. ns: non-significant.

FIGURE 2. Maturation of the TEC subsets defined by Ly51/UEA1 expression during fetal development (E12.5-E17.5) in both WT and EphB-deficient mice. (A) Dot plots, representative of the performed analyses gating in total WT and mutant EpCAM⁺CD45⁻ epithelial cells, show the maturation of distinct cell subsets defined by Ly51 and/or UEA1 expression. (B) Proportions of both WT and mutant thymic Ly51⁻UEA1⁻ cells throughout embryonic development. (C) Proportions of both WT and mutant thymic Ly51^loUEA1⁻ cells during development. (D) Changes in the proportions of thymic Ly51^medUEA1⁻ cells in WT and mutant embryonic thymuses. Proportions of both Ly51^medUEA1^lo/med (E) and Ly51^hiUEA1^lo/med (F) cell subsets during embryonic development of WT and mutant thymuses. The significance of the Student's t-test probability is indicated as *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.005; or #p ≤ 0.05; ###p ≤ 0.005. ns: non-significant.

FIGURE 3. Maturation of cTEC subsets based on Ly51/CD205 expression in both WT and EphB-deficient thymuses. (A) Dot plots show the evolution of Ly51⁺CD205⁻ and Ly51⁺CD205⁺ cTEC subpopulations at E14.5 and E15.5 in both WT and mutant thymuses. (B) Proportions of both Ly51⁺CD205⁻ and Ly51⁺CD205⁺ cTECs in WT and mutant thymuses. The significance of the Student's t-test probability is indicated as *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.005; or #p ≤ 0.05; ##p ≤ 0.01; ###p ≤ 0.005. ns: non-significant.

FIGURE 4. cTEC subsets defined by the expression of Ly51 and MHCII cell markers during fetal development (E12.5-E17.5) of both WT and EphB-deficient thymuses. (A) Dot plots show different TEC subsets defined by Ly51/MHCII expression throughout thymus development. They are representative of different analyses of TEC subsets gated in the total EpCAM⁺CD45⁻ epithelial cell population. (B) Proportions of MHCII⁻Ly51⁻ cells in WT and EphB-deficient embryonic thymuses. Proportions of both MHCII^loLy51^lo (C) and MHCII^medLy51^lo (D) cells in WT and mutant thymuses. (E) Proportions of MHCII^medLy51^med cells in WT and mutant embryonic thymuses. (F, G) Percentage of mature MHCII^hiLy51^med (F) and MHCII^hiLy51^hi (G) cTECs in WT and mutant embryonic thymuses. The significance of the Student's t-test probability is indicated as *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.005 or #p ≤ 0.05; ##p ≤ 0.01; ###p ≤ 0.005. ns: non-significant.

FIGURE 5. cTEC subsets defined by the expression of Ly51 and CD40 or β5t cell markers and epithelial cell apoptosis during fetal development in both WT and EphB-deficient mice. (A) Dot plots show different TEC subsets defined by CD40/Ly51 expression throughout thymus development. CD40 expression in thymic cortex defines three cell subsets: negative (−), low (^lo) (B, C) and medium/high (^med/hi) (D, E) whose maturation in WT and mutant thymuses is represented in figures A to E. (F) The histogram shows a representative example of β5t expression (black line) in cortical EpCAM⁺CD45⁻Ly51⁺ cells (R1) at E15.5 respect to the negative expression in thymocytes (grey line). (G) Proportions of β5t⁺ cells in the cTEC subset, at E13.5, E15.5 and E17.5, determined by flow cytometry. (H) Significantly lower proportions of apoptotic EpCAM⁺CD45⁻ epithelial cells at E12.5 and higher at E13.5 in mutant thymuses. The significance of the Student's t-test probability is indicated as *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.005 or #p ≤ 0.05; ##p ≤ 0.01; ###p ≤ 0.005. ns: non-significant.

FIGURE 6. Proportions of cycling cells in different epithelial cell subsets of both WT and EphB-mutant thymuses during fetal development (E12.5-E17.5). Figure represents the proportions of cycling cells defined as cells in S⁺G2/M phases. (A) Proportions of cycling total EpCAM⁺CD45⁻ cells in both WT and mutant. (B) Changes in the proportions of cycling MTS20⁺ cells during thymus development are similar to those observed for the total EpCAM⁺CD45⁻ TECs in both WT and mutant thymuses. (C) Proportions of both WT and mutant cycling MTS20⁻ cells during thymus maturation. (D) Proportions of cycling Ly51⁻UEA1⁻ cells in WT and mutant developing thymuses. (E) Percentage of cycling Ly51⁺UEA1⁻ cTECs in mutant and WT thymuses. The significance of the Student's t-test probability is indicated as *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.005 or #p ≤ 0.05; ##p ≤ 0.01; ###p ≤ 0.005. ns: non-significant.

FIGURE 7. Expression of EphB and ephrin-B in WT thymic cells during early development (E12.5-E15.5). The figure shows the proportions of either TECs (A) or thymocytes (B) expressing EphB2, EphB3, ephrin-B1 and ephrin-B2 evaluated by flow cytometry. (C) Pattern of expression of EphB and ephrin-B in the WT and mutant Ly51⁻UEA1⁻ cell population (D) Variations in the proportions of Eph/ephrin-B-expressing Ly51⁺UEA1⁻ cTECs in WT and mutant embryonic thymuses. The significance of the Student's t-test probability is indicated between stages as *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.005.

FIGURE 8. Expression of genes involved in thymic cell maturation in EphB-deficient thymuses. Figure shows the relative expression, RQ (2⁻ΔΔCt), of FoxN1, FGF7, FGFR2IIIb, IL7 and Dll4 transcripts in mutant cells relative to WT values (value 1, dotted line). At E12.5, the expression was determined on total thymic lobes whereas at E15.5 sorted EpCAM⁺CD45⁻ TECs or, for FGF7 expression, EpCAM⁻CD45⁻ mesenchymal cells were studied. The relative expression of mutant values was compared to the WT using the one-t test and between mutants according to Student's t-test. The significance is indicated as: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.005 or #p ≤ 0.05; ##p ≤ 0.01. ns: non-significant.