Figure 4
The propensity of P2neo/neo thymocytes to undergo apoptosis is associated with elevated expression of TCRβ and TCRγδ. (A and B) Expression of TCRβ and TCRγδ on WT and P2neo/neo embryonal thymocytes. (A) Shown are FACS analysis dot plots of E15.5 to E18.5 WT and P2neo/neo thymocytes indicating the percentages of cells in each quadrant. Histograms in (B) show the average ± S.E. of TCRβ/TCRγδ proportions of WT and P2neo/neo thymocytes in at least three experiments at each developmental stage. The difference between WT and P2neo/neo was significant at P < 0.005 (*) and P < 0.05 (#) by Student's t test. While at E15.5 to E17.5 the proportion of TCRβ/TCRγδ expressing thymocytes was much higher in P2neo/neo compared to WT, at E18.5 the distribution of P2neo/neo thymocytes resumed normal pattern. The mean fluorescence intensity (MFI) (B right) of E15.5 and E16.5 TCRβ+ P2neo/neo thymocytes was significantly higher than WT (p < 0.005; by Student's t test). (C) Expression of TCRβ/TCRγδ in P2neo/neo thymocytes is transcriptionally upregulated. RT-PCR analysis of RNA derived from WT and P2neo/neo E16.5 thymocytes using primers specific for the pre-TCRα and the constant regions of TCRβ and TCRγ transcripts. Increased number of PCR cycles shows elevated steady-state levels of mRNAs in P2neo/neo thymocytes compared to WT. (D and E) Enhanced apoptosis of P2neo/neo thymocytes is associated with elevated expression of TCRβ/TCRγδ. (D) Histograms demonstrating the proportion of apoptotic cells among TCRβ+ or TCRgδ+ thymocytes. TCRβ/TCRγδ positive WT thymocytes (blue) are divided into two main populations, apoptotic (M1) and non-apoptotic, according to level of Annexin V (except for E15.5 TCRγδ+ where all cells are apoptotic). Most of TCRβ/TCRγδ positive P2neo/neo thymocytes (red), are at the M1 (Annexin Vhigh) apoptotic subset. (E) Proportion of apoptotic thymocytes among TCRβ/TCRγδ positive E15.5, E16.5 and E18.5 thymocytes. Bars represent the average ± S.E. of at least three independent experiments for each time point using different mice. The differences between WT and P2neo/neo in number of TCRβ+ apoptotic thymocytes were significant at P < 0.01 (*) by Student's t test.