Figure 4.

V(D)J recombination defect and impaired development of thymocytes in PAXX^−/−/Xlf^−/− E18.5 embryos. (a) Immunostaining of thymocytes from wt, Xlf^−/−, PAXX^−/−, and PAXX^−/−Xlf^−/− E18.5 embryos. Upper panel (red) shows the important decrease in CD4⁺CD8⁺ DP in the thymus from PAXX^−/−Xlf^−/− embryos. DN (CD4⁻CD8⁻CD3⁻) thymocytes were subdivided into DN1–DN4 according to the expression of CD44 and CD25 (middle panel). Lower panel (green) shows the severe block at the transition DN3a (CD44⁻CD25⁺CD28⁻) to DN3b (CD44⁻CD25⁺CD28⁺) in PAXX^−/−Xlf^−/− embryos. B220⁺CD19⁺ FL cells were analyzed for the expression of CD43 that marks pro-B cells (middle panel). In the lower panel, cells were gated on the CD43⁺B220⁺CD19⁺ population and analyzed for iIgM expression. (b) Quantification of thymocyte subpopulations in the various PAXX/Xlf genotypes. Mann–Whitney test was used for statistical analysis. (c) PCR strategy to analyze TCRβ rearrangement according to Gartner et al.²⁹ and autoradiogram of PCR products revealed with the TCR-Jβ probe demonstrating the absence of either Dβ-Jβ or VβDβJβ rearrangements in thymocytes from PAXX^−/−Xlf^−/− embryos. GAPDH-specific PCR was used as loading control