Ablation in embryonic thymocyte development. E18.5 thymocytes were analyzed by flow cytometry. A. DN thymocytes were gated on CD45+Lin2CD32CD42CD82 cells. Results show percentage of DN1 N4 in Ret, Gfra1and Gfra2 deficient mice. Null mice: open symbols; WT littermate controls: full symbols; Mean value: dash line. B. Percentage of DN and DP thymocytes gated on CD45+Lin2cdTCR2 analyzed as in Figure 2A. C. Percentage of cd TCR expressing thymocytes analyzed as in Figure 2A. D. Absolute number of total thymocytes in Ret,RET MedChemExpress KS-176 signalling and T Cell DevelopmentGfra1and Gfra2 deficient mice analyzed as in Figure 
2A. Two-tailed student t-test analysis was performed between knockouts and respective WT littermate controls. No statistically significant differences were found. doi:10.1371/journal.pone.0052949.gsignalling is dispensable for thymocyte competitive fitness and thymic reconstitution.DiscussionOur data indicate that the neuroregulatory genes Ret, Gfra1 and Gfra2 are expressed in discrete DN thymocytes, while Gfra3 and Gfra4 transcripts were absent in thymocytes. Interestingly, the RET ligands Gdnf and Nrtn are predominantly produced by nonhematopoietic thymic cells. These gene Docosahexaenoyl ethanolamide web expression patterns raised the exciting possibility that RET signalling axes could control T cell development, adding to the growing body of evidence that the nervous and immune systems share similar key molecular signals [11,14,17,18,19,25]. In line with this hypothesis, it was previously shown that GDNF could promote survival and maturation of thymocytes in vitro [11]. In order to test whether RET signalling axes control T cell development, we analyzed the thymus of genetically mutant embryos for Ret, Gfra1 or Gfra2 at E18.5 [20,21,22]. Despite expression of Ret in foetal thymocytes, Ret deficient embryos showed normal DN, immCD8 DP and cd T cell development. These findings were also consistent with normal T cell development in null mice for the RET co-receptors that provide specificity to the neurotrophic factors GDNF and NRTN, respectively Gfra12/2 and Gfra22/2 mice. Thus, we conclude that RET and its signalling partners GFRa1 and GFRa2, are dispensable for foetal T cell development.Since T cell development in adulthood employs additional molecular mechanisms to foetal thymopoiesis, we investigated whether Ret related genes controlled adult T cell development. Our data indicate co-expression of Ret, Gfra1 and Gfra2 in the early DN1 stage, and production of Gdnf and Nrtn in the adult thymic microenvironment. Both foetal and adult immature thymocytes co-express Ret, Gfra1 and Gfra2. These data are in line with a previous report indicating expression of these genes in the foetal thymus by in situ hybridization [10]. However, DP thymocytes expressed the RET co-receptors Gfra1 and Gfra2, despite absence of Ret expression, and similarly, adult DN2? expressed Gfra1 but lacked significant Ret expression. This observation is in line with previous reports showing that GFRas are more abundantly expressed than RET [26,27], which suggests that GFRas may modulate RET signalling in a non-cell-autonomous manner (signalling in trans) [28,29]. Accordingly, we have recently shown that Lymphoid Tissue initiator cells use unconventional RET signalling in which receptor activation is provided by soluble ligand and co-receptors in trans secreted from nearby cells [17]. Thus, it is possible that RET negative, GFRa positive thymocytes may modulate the activity of neighbouring non-hematop.Ablation in embryonic thymocyte development. E18.5 thymocytes were analyzed by flow cytometry. A. DN thymocytes were gated on CD45+Lin2CD32CD42CD82 cells. Results show percentage of DN1 N4 in Ret, Gfra1and Gfra2 deficient mice. Null mice: open symbols; WT littermate controls: full symbols; Mean value: dash line. B. Percentage of DN and DP thymocytes gated on CD45+Lin2cdTCR2 analyzed as in Figure 2A. C. Percentage of cd TCR expressing thymocytes analyzed as in Figure 2A. D. Absolute number of total thymocytes in Ret,RET Signalling and T Cell DevelopmentGfra1and Gfra2 deficient mice analyzed as in Figure 2A. Two-tailed student t-test analysis was performed between knockouts and respective WT littermate controls. No statistically significant differences were found. doi:10.1371/journal.pone.0052949.gsignalling is dispensable for thymocyte competitive fitness and thymic reconstitution.DiscussionOur data indicate that the neuroregulatory genes Ret, Gfra1 and Gfra2 are expressed in discrete DN thymocytes, while Gfra3 and Gfra4 transcripts were absent in thymocytes. Interestingly, the RET ligands Gdnf and Nrtn are predominantly produced by nonhematopoietic thymic cells. These gene expression patterns raised the exciting possibility that RET signalling axes could control T cell development, adding to the growing body of evidence that the nervous and immune systems share similar key molecular signals [11,14,17,18,19,25]. In line with this hypothesis, it was previously shown that GDNF could promote survival and maturation of thymocytes in vitro [11]. In order to test whether RET signalling axes control T cell development, we analyzed the thymus of genetically mutant embryos for Ret, Gfra1 or Gfra2 at E18.5 [20,21,22]. Despite expression of Ret in foetal thymocytes, Ret deficient embryos showed normal DN, immCD8 DP and cd T cell development. These findings were also consistent with normal T cell development in null mice for the RET co-receptors that provide specificity to the neurotrophic factors GDNF and NRTN, respectively Gfra12/2 and Gfra22/2 mice. Thus, we conclude that RET and its signalling partners GFRa1 and GFRa2, are dispensable for foetal T cell development.Since T cell development in adulthood employs additional molecular mechanisms to foetal thymopoiesis, we investigated whether Ret related genes controlled adult T cell development. 
Our data indicate co-expression of Ret, Gfra1 and Gfra2 in the early DN1 stage, and production of Gdnf and Nrtn in the adult thymic microenvironment. Both foetal and adult immature thymocytes co-express Ret, Gfra1 and Gfra2. These data are in line with a previous report indicating expression of these genes in the foetal thymus by in situ hybridization [10]. However, DP thymocytes expressed the RET co-receptors Gfra1 and Gfra2, despite absence of Ret expression, and similarly, adult DN2? expressed Gfra1 but lacked significant Ret expression. This observation is in line with previous reports showing that GFRas are more abundantly expressed than RET [26,27], which suggests that GFRas may modulate RET signalling in a non-cell-autonomous manner (signalling in trans) [28,29]. Accordingly, we have recently shown that Lymphoid Tissue initiator cells use unconventional RET signalling in which receptor activation is provided by soluble ligand and co-receptors in trans secreted from nearby cells [17]. Thus, it is possible that RET negative, GFRa positive thymocytes may modulate the activity of neighbouring non-hematop.