Fra1and Gfra2 deficient mice. B. Dot plots: CD4 and CD8 expression profiles within the CD45+LinnegcdTCR2 compartment from an example of Ret2/2and respective WT littermate controls. Similar gates were used in results shown. Note that within SPCD4 and SPCD8 gates .90 of cells were CD32 and are thus immature thymocytes. Results show percentage and absolute numbers of immature CD8+ thymocytes and absolute numbers of DN and DP thymocytes in Ret, Gfra1and Gfra2 deficient mice. C. Absolute numbers of cd TCR+ thymocytes in Ret, Gfra1and Gfra2 deficient mice. In all panels: Null mice: open symbols; WT littermate controls: full symbols; Mean value: dash line. Two-tailed student ttest analysis was performed between knockouts and respective WT littermate controls. No statistically significant differences were found. (TIF) Figure S2 Generation of Ret conditional knockout mice. A. Adult (8 weeks old) DN, DP, single-positive CD8 (SP8) and single-positive CD4 (SP4) thymocytes were purified by flow cytometry. RT-PCR analysis was performed. B. (A) The floxed Neomycin cassette was inserted ,4.5 kb upstream of exon 1 of mouse Ret locus, a third loxP (LoxP3) 
was introduced downstream of exon 1 and ,5 kb downstream the PGK-TK-pA cassette was inserted to aid negative selection. Targeted events were A196 custom synthesis identified by Southern analysis of either Hind III digests of genomic DNA using the 59 external probe. (B) The floxed allele was identified by PCR and the primers P1/P2 were used to identify the loxP that remained after excision of the Neomycin cassette (PGK-Neo-PA), while the loxP3 was identified using primers P3/P4. The primer sequences are in the methods section. (C) To screen for the null allele, primers P1 and P4 were used. C. Genotyping results from a litter of mice obtained from a cRet131WT/null6cRet131fl/fl breeding. 23977191 In the loxP sites PCRs, upper band corresponds to the sequence with the loxP site and the lower band to the WTFlow cytometryEmbryonic thymi were micro-dissected and either homogenized in 70 mM cell strainers or Bromopyruvic acid digested with Accutase medium (PAA Laboratories, Austria), 309 at 37u. Adult thymi were homogenized in 70 mm cell strainers. Single cell suspensions were stained with the following antibodies from ebioscience, Biolegend, or BD: antiCD3 (145-2C11), anti-CD4 (RM4-5), anti-CD8 (53-6.7), antiCD44 (IM7), anti-CD25 (7D4), anti-CD45 (30-F11), anti-CD45.1 (A20), antiCD45.2 (104), anti-cd TCR (GL3), anti-CD117 (2B8), anti-Sca1 (D7), Lineage (Lin) cocktail (anti-CD19 (eBio1D3), antiCD11b (M1/70), anti-Gr.1 (RB6-8C5), anti-Ly79 (Ter119) and anti-NK1.1 (PK136)). Antibodies were coupled to FITC, PE, PerCP, PerCP-Cy5, PE-Cy7, APC, APC-Cy7, Pacific Blue, Brilliant Violet 421 and Horizon V500 fluorochromes or to biotin. Secondary incubation with fluorochrome binding 
streptavidin was performed when biotin coupled antibodies were used. Anti hRET was performed with antibody from R D (132507) and respective anti-mouse IgG1 isotype control. Flow cytometry analysis was performed on a LSR Fortessa (BD) and data was analyzed with FlowJo 8.8.7 software (Tree Star). Cell-sorting was performed on a FACSAria I or FACSAria III (BD), and purity of obtained samples was .97 . CD45+ and CD452 populations were sorted from the same samples.Real-time PCR analysisRNA was extracted from sorted cell suspensions using RNeasy Micro Kit (Qiagen). RT-PCR was performed as previously described [18] and quantitative Real-time PCR for Gfra1 and Gfra2 were done as previously describe.Fra1and Gfra2 deficient mice. B. Dot plots: CD4 and CD8 expression profiles within the CD45+LinnegcdTCR2 compartment from an example of Ret2/2and respective WT littermate controls. Similar gates were used in results shown. Note that within SPCD4 and SPCD8 gates .90 of cells were CD32 and are thus immature thymocytes. Results show percentage and absolute numbers of immature CD8+ thymocytes and absolute numbers of DN and DP thymocytes in Ret, Gfra1and Gfra2 deficient mice. C. Absolute numbers of cd TCR+ thymocytes in Ret, Gfra1and Gfra2 deficient mice. In all panels: Null mice: open symbols; WT littermate controls: full symbols; Mean value: dash line. Two-tailed student ttest analysis was performed between knockouts and respective WT littermate controls. No statistically significant differences were found. (TIF) Figure S2 Generation of Ret conditional knockout mice. A. Adult (8 weeks old) DN, DP, single-positive CD8 (SP8) and single-positive CD4 (SP4) thymocytes were purified by flow cytometry. RT-PCR analysis was performed. B. (A) The floxed Neomycin cassette was inserted ,4.5 kb upstream of exon 1 of mouse Ret locus, a third loxP (LoxP3) was introduced downstream of exon 1 and ,5 kb downstream the PGK-TK-pA cassette was inserted to aid negative selection. Targeted events were identified by Southern analysis of either Hind III digests of genomic DNA using the 59 external probe. (B) The floxed allele was identified by PCR and the primers P1/P2 were used to identify the loxP that remained after excision of the Neomycin cassette (PGK-Neo-PA), while the loxP3 was identified using primers P3/P4. The primer sequences are in the methods section. (C) To screen for the null allele, primers P1 and P4 were used. C. Genotyping results from a litter of mice obtained from a cRet131WT/null6cRet131fl/fl breeding. 23977191 In the loxP sites PCRs, upper band corresponds to the sequence with the loxP site and the lower band to the WTFlow cytometryEmbryonic thymi were micro-dissected and either homogenized in 70 mM cell strainers or digested with Accutase medium (PAA Laboratories, Austria), 309 at 37u. Adult thymi were homogenized in 70 mm cell strainers. Single cell suspensions were stained with the following antibodies from ebioscience, Biolegend, or BD: antiCD3 (145-2C11), anti-CD4 (RM4-5), anti-CD8 (53-6.7), antiCD44 (IM7), anti-CD25 (7D4), anti-CD45 (30-F11), anti-CD45.1 (A20), antiCD45.2 (104), anti-cd TCR (GL3), anti-CD117 (2B8), anti-Sca1 (D7), Lineage (Lin) cocktail (anti-CD19 (eBio1D3), antiCD11b (M1/70), anti-Gr.1 (RB6-8C5), anti-Ly79 (Ter119) and anti-NK1.1 (PK136)). Antibodies were coupled to FITC, PE, PerCP, PerCP-Cy5, PE-Cy7, APC, APC-Cy7, Pacific Blue, Brilliant Violet 421 and Horizon V500 fluorochromes or to biotin. Secondary incubation with fluorochrome binding streptavidin was performed when biotin coupled antibodies were used. Anti hRET was performed with antibody from R D (132507) and respective anti-mouse IgG1 isotype control. Flow cytometry analysis was performed on a LSR Fortessa (BD) and data was analyzed with FlowJo 8.8.7 software (Tree Star). Cell-sorting was performed on a FACSAria I or FACSAria III (BD), and purity of obtained samples was .97 . CD45+ and CD452 populations were sorted from the same samples.Real-time PCR analysisRNA was extracted from sorted cell suspensions using RNeasy Micro Kit (Qiagen). RT-PCR was performed as previously described [18] and quantitative Real-time PCR for Gfra1 and Gfra2 were done as previously describe.
