Figure 3
P2neo/neo thymocytes display enhanced apoptosis, but have normal cell proliferation capacity. (A) FACS analysis of E14.5 and E17.5 WT (left panels) and P2neo/neo (right panels) thymocytes stained with Annexin V and PI. FACS dot plots and percentages of cells in each quadrant are indicated. Note the >2 fold increase in the proportion of apoptotic cells (Annexin V+/PI-) and of necrotic cells (Annexin V+/PI+) in P2neo/neo thymi. (B) P2neo/neo thymocytes display enhanced apoptosis throughout embryonic thymopoiesis. Proportion of non-viable (apoptotic+necrotic) WT and P2neo/neo cells gradually decreased during embryonic development. Nevertheless, at any given time point, P2neo/neo thymui contained a higher proportion of non-viable cells compared to WT littermates. After birth the proportion of non-viable thymocytes in P2neo/neo became similar to WT. At least five mice of each genotype were analyzed. The differences between WT and P2neo/neo apoptotic thymocytes were significant at P < 0.001 (*) and P < 0.01 (#) by Student's t test. (C) P2neo/neo thymocytes retain normal proliferation capacity. Following FACS analysis an equal number (1 × 104) of E15.5 WT or P2neo/neo Annexin V negative thymocytes were incubated with TPA (20 ng/ml) and ConA (5 μg/ml) for 48 h. 3H-thymidine was present during the last 18 h of incubation. Differences between WT and P2neo/neo were statistically insignificant by Student's t test. Proliferation capability of thymocytes was further examined by immunostaining of E15.5 thymic lobes for the proliferation-associated antigen recognized by Ki-67 antibodies (Right panels). (D) Enhanced embryonal apoptosis is a cell-autonomous property of P2neo/neo thymocytes. WT or P2neo/neo E15.5 FL HPC were differentiated under saturating conditions in E14.5 WT FTOC for 14 or 16 days. We predetermined that ~100 FL cells (either WT or P2neo/neo) per lobe were sufficient to populate all available thymic lobes and therefore used 1000 FL cells per lobe. Thymocytes accumulation in FTOC populated with P2neo/neo FL HPC is reduced compared to WT (left panel WT-black; P2neo/neo-white). Proportion of apoptotic thymocytes (Annexin V+) derived from FTOC populated with P2neo/neo FL cells (red) was considerably higher compared to WT (light blue) (right panel).