Ivo, since Ret mutant thymocytes JW-74 web 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 57773-65-6 web 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 (open squares) and WT control (full circle) mice. Mean value: dash line. C. Proportion and absolute numbers of cd TCR expressing thymocytes in MEN2B (open squares) and WT control (full circle) mice. Mean value: dash line. D. Absolute thymocyte numbers. Two-tailed student t-test analysis was performed between knockouts and respective controls. No statistically significant differences were found. doi:10.1371/journal.pone.0052949.gcell development in vivo appears to be insignificant. Moreover, while FTOCs reproduce several aspects of T cell development [30], they fail to mimic the exact events in T cell development [31,32], and therefore these different methodologies may also contribute to the observed discrepancies.GDNF/GFRa1 have been shown to activate the transmembrane receptor RET and the neural cell adhesion molecule (NCAM) in neurons [33,34]. Thus, although activation of a putative NCAM analogue by GDNF cannot be fully discarded in thymocytes, this is unlikely to have a significant physiologicalRET Signalling and T Cell DevelopmentFigure 6. Competitive fitness and thymic reconstitution of Ret-null thymocytes. A. Experimental scheme: 9Gy irradiated hosts (Rag12/2, CD45.1) received WT competitor precursors (CD45.1/2) together with hCD2Cre/Retnull/fl or control hCD2Cre2/Retwt/fl precursors (CD45.2). B. 8 weeks after transplantation the thymus of the generated chimeras was analyzed by flow cytometry. Results show the ratio between hCD2Cre/Retnull/fl (grey bar) or hCD2Cre2/Retwt/fl (black bar) and the third part WT competitor (CD45.1/2) through thymic T cell development. hCD2Cre/Retnull/fl precursor chimeras: n = 4; hCD2Cre2/Retwt/fl precursor chimeras n = 4. Error bars show s.d. Two-tailed student t-tests were performed. No significant differences were found. doi:10.1371/journal.pone.0052949.grelevance since NCAM downstream signalling requires GFRa1 and Gfra12/2 embryos displayed normal thymopoiesis [33]. In order to overcome possible viability/proliferative compensatory mechanisms that may arise through T cell development, we performed sensitive competitive reconstitution assays in vivo with Ret deficient (CD2Cre/Retnull/fl) and Ret competent (CD2Cre/ RetWT/fl) thymocytes. Our data demonstrate that even in a very sensitive competitive 11967625 setting the fitness of Ret deficient T cell precursors is intact. Finally, our findings indicate that pharmacological inhibition of the RET pathway in severe pathologies, such as medullary thyroid cancer, should not be confronted with undesirable T cell production failure [15,16]. In summary, our data demonstrate that RET signalling is dispensable to foetal and adult T cell development in vivo. Nevertheless, RET.Ivo, 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 (open squares) and WT control (full circle) mice. Mean value: dash line. C. Proportion and absolute numbers of cd TCR expressing thymocytes in MEN2B (open squares) and WT control (full circle) mice. Mean value: dash line. D. Absolute thymocyte numbers. Two-tailed student t-test analysis was performed between knockouts and respective controls. No statistically significant differences were found. doi:10.1371/journal.pone.0052949.gcell development in vivo appears to be insignificant. Moreover, while FTOCs reproduce several aspects of T cell development [30], they fail to mimic the exact events in T cell development [31,32], and therefore these different methodologies may also contribute to the observed discrepancies.GDNF/GFRa1 have been shown to activate the transmembrane receptor RET and the neural cell adhesion molecule (NCAM) in neurons [33,34]. Thus, although activation of a putative NCAM analogue by GDNF cannot be fully discarded in thymocytes, this is unlikely to have a significant physiologicalRET Signalling and T Cell DevelopmentFigure 6. Competitive fitness and thymic reconstitution of Ret-null thymocytes. A. Experimental scheme: 9Gy irradiated hosts (Rag12/2, CD45.1) received WT competitor precursors (CD45.1/2) together with hCD2Cre/Retnull/fl or control hCD2Cre2/Retwt/fl precursors (CD45.2). B. 8 weeks after transplantation the thymus of the generated chimeras was analyzed by flow cytometry. Results show the ratio between hCD2Cre/Retnull/fl (grey bar) or hCD2Cre2/Retwt/fl (black bar) and the third part WT competitor (CD45.1/2) through thymic T cell development. hCD2Cre/Retnull/fl precursor chimeras: n = 4; hCD2Cre2/Retwt/fl precursor chimeras n = 4. Error bars show s.d. Two-tailed student t-tests were performed. No significant differences were found. doi:10.1371/journal.pone.0052949.grelevance since NCAM downstream signalling requires GFRa1 and Gfra12/2 embryos displayed normal thymopoiesis [33]. In order to overcome possible viability/proliferative compensatory mechanisms that may arise through T cell development, we performed sensitive competitive reconstitution assays in vivo with Ret deficient (CD2Cre/Retnull/fl) and Ret competent (CD2Cre/ RetWT/fl) thymocytes. Our data demonstrate that even in a very sensitive competitive 11967625 setting the fitness of Ret deficient T cell precursors is intact. Finally, our findings indicate that pharmacological inhibition of the RET pathway in severe pathologies, such as medullary thyroid cancer, should not be confronted with undesirable T cell production failure [15,16]. In summary, our data demonstrate that RET signalling is dispensable to foetal and adult T cell development in vivo. Nevertheless, RET.
