Oietic RET expressing cells. Analysis of adult conditional Ret mutant mice (Retfl/fl) bred to CD2Cre mice revealed a significant, but small impact on the absolute numbers of DN1 thymocytes. However, this reduction was not translated into consecutive developmental stages, indicating that RET mediated signals are dispensable to T cellFigure 3. Expression of Ret and its signalling partners in adult thymic populations. A. DN, DP, SPCD8+ and SPCD4+ thymocytes were purified by flow cytometry. Results show quantitative RT-PCR normalized to Hprt1. Error bars show s.e.. Results from three independent measurements 15900046 are represented. B. 
RET expression in DN and DP thymocytes was determined by flow cytometry. RET: black bold line; Isotype control: grey line. C. Thymic DN, DP, SPCD8+ and SPCD4+ thymocytes and CD452 cells were purified by flow cytometry. Quantitative RT-PCR analysis was normalized to Hprt1. Error bars show s.e.. Results from three independent measurements are represented. D. DN1? and DN3? thymocytes were purified by flow cytometry. Results show quantitative RT-PCR normalized to Hprt1. Error bars show s.e.. Results from three independent measurements are represented. doi:10.1371/journal.pone.0052949.gRET Signalling and T Cell DevelopmentFigure 4. Impact of Ret ablation in adult thymic development. 8 week old Ret conditional knockout hCD2Cre/Retnull/fl and control hCD2Cre2/ Retwt/fl mice were TA01 site analyzed by flow cytometry. A. Left: representative flow cytometry analysis of I-BRD9 site CD42CD82CD32 thymocytes. Percentages are indicated. Right: Results show percentage of DN1-DN4 in hCD2Cre/Retnull/fl (open circle) and control hCD2Cre2/Retwt/fl (full circle) mice. Mean value: dash line. B. Left: representative flow cytometry analysis of CD4 versus CD8 expression profile. Percentages are indicated. Right: Results show percentage of DN, DP, SP4 and SP4 in hCD2Cre/Retnull/fl (open circle) and control hCD2Cre2/Retwt/fl (full circle) mice. 
Mean value: dash line. C. Proportion and absolute numbers of cd TCR expressing thymocytes in hCD2Cre/Retnull/fl (open circle) and control hCD2Cre2/Retwt/fl (full circle) mice. Mean value: dash line. D. Absolute thymocyte numbers. Two-tailed student t-test analysis was performed between mutant and respective control mice. No statistically significant differences were found. doi:10.1371/journal.pone.0052949.gproduction in vivo. The co-expression of Ret/Gfra1 in DN1 and the decreased number of DN1 cells in Retfl/fl mice are consistent with a previous report indicating that GDNF promotes thymocyte survival in foetal thymic organ cultures (FTOCs) [11]. Neverthe-less, our data does not support a major role for this signalling axis in vivo, since Ret mutant thymocytes develop normally and positive modulation of RET-signalling (RetMEN2B) was not beneficial for T cell development. Thus, the contribution of RET signalling to TRET Signalling and T Cell DevelopmentFigure 5. RetMEN2B gain-of-function mutation in adult thymocyte development. 8 week old RetMEN2B/MEN2B (MEN2B) and their WT littermate controls were analyzed by flow cytometry. A. Left: representative flow cytometry analysis of CD42CD82CD32 thymocytes. Percentages are indicated. Right: Results show percentage of DN1 N4 in MEN2B (open squares) and WT control (full circle) mice. Mean value: dash line. B. Left: representative flow cytromety analysis of CD4 versus CD8 expression profile. Percentages are indicated. Right: Results show percentage of DN, DP, SP4 and SP4 in in MEN2B (.Oietic RET expressing cells. Analysis of adult conditional Ret mutant mice (Retfl/fl) bred to CD2Cre mice revealed a significant, but small impact on the absolute numbers of DN1 thymocytes. However, this reduction was not translated into consecutive developmental stages, indicating that RET mediated signals are dispensable to T cellFigure 3. Expression of Ret and its signalling partners in adult thymic populations. A. DN, DP, SPCD8+ and SPCD4+ thymocytes were purified by flow cytometry. Results show quantitative RT-PCR normalized to Hprt1. Error bars show s.e.. Results from three independent measurements 15900046 are represented. B. RET expression in DN and DP thymocytes was determined by flow cytometry. RET: black bold line; Isotype control: grey line. C. Thymic DN, DP, SPCD8+ and SPCD4+ thymocytes and CD452 cells were purified by flow cytometry. Quantitative RT-PCR analysis was normalized to Hprt1. Error bars show s.e.. Results from three independent measurements are represented. D. DN1? and DN3? thymocytes were purified by flow cytometry. Results show quantitative RT-PCR normalized to Hprt1. Error bars show s.e.. Results from three independent measurements are represented. doi:10.1371/journal.pone.0052949.gRET Signalling and T Cell DevelopmentFigure 4. Impact of Ret ablation in adult thymic development. 8 week old Ret conditional knockout hCD2Cre/Retnull/fl and control hCD2Cre2/ Retwt/fl mice were analyzed by flow cytometry. A. Left: representative flow cytometry analysis of CD42CD82CD32 thymocytes. Percentages are indicated. Right: Results show percentage of DN1-DN4 in hCD2Cre/Retnull/fl (open circle) and control hCD2Cre2/Retwt/fl (full circle) mice. Mean value: dash line. B. Left: representative flow cytometry analysis of CD4 versus CD8 expression profile. Percentages are indicated. Right: Results show percentage of DN, DP, SP4 and SP4 in hCD2Cre/Retnull/fl (open circle) and control hCD2Cre2/Retwt/fl (full circle) mice. Mean value: dash line. C. Proportion and absolute numbers of cd TCR expressing thymocytes in hCD2Cre/Retnull/fl (open circle) and control hCD2Cre2/Retwt/fl (full circle) mice. Mean value: dash line. D. Absolute thymocyte numbers. Two-tailed student t-test analysis was performed between mutant and respective control mice. No statistically significant differences were found. doi:10.1371/journal.pone.0052949.gproduction in vivo. The co-expression of Ret/Gfra1 in DN1 and the decreased number of DN1 cells in Retfl/fl mice are consistent with a previous report indicating that GDNF promotes thymocyte survival in foetal thymic organ cultures (FTOCs) [11]. Neverthe-less, our data does not support a major role for this signalling axis in vivo, since Ret mutant thymocytes develop normally and positive modulation of RET-signalling (RetMEN2B) was not beneficial for T cell development. Thus, the contribution of RET signalling to TRET Signalling and T Cell DevelopmentFigure 5. RetMEN2B gain-of-function mutation in adult thymocyte development. 8 week old RetMEN2B/MEN2B (MEN2B) and their WT littermate controls were analyzed by flow cytometry. A. Left: representative flow cytometry analysis of CD42CD82CD32 thymocytes. Percentages are indicated. Right: Results show percentage of DN1 N4 in MEN2B (open squares) and WT control (full circle) mice. Mean value: dash line. B. Left: representative flow cytromety analysis of CD4 versus CD8 expression profile. Percentages are indicated. Right: Results show percentage of DN, DP, SP4 and SP4 in in MEN2B (.
