D [18,36]. Hprt1 was used as housekeeping gene. For TaqMan assays (Applied Biosystems) RNA was retro-transcribed using High Capacity RNA-to-cDNA Kit (Applied Biosystems), followed by a pre-amplification PCR using TaqMan PreAmp Master Mix (Applied Biosystems). TaqRET Signalling and T Cell Developmentsequence. D. In order to evaluate the activity of Cre recombinase driven by hCD2, we bred hCD2Cre-expressing animals to Rosa26 eYFP animals. Histograms show flow cytometry analysis of eYFP expression in DN1 to DN4 thymocytes. (TIF)Figure S3 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. Results show absolute numbers of DN1 N4 (top) and DN to mature single MK 8931 web positive (bottom) in hCD2Cre/Retnull/fl (open circle) and control hCD2Cre2/Retwt/fl (full circle) mice. Mean value: dash line. All WT and conditional Ret knockout deficient pairs were compared using two-tailed student t-tests, and no significant differences were found except where noted. 
*p,0.05. (TIF) Figure S4 Impact of Ret gain-of-function mutation RetMEN2B in adult thymic development. 8 week old RetMEN2B/MEN2B (MEN2B) and their WT littermate NT 157 supplier controls wereanalyzed by flow cytometry. Results show absolute numbers of DN1 N4 (top) and DN to mature SP (bottom) in MEN2B (open squares) and WT control (full circle) mice. Mean value: dash line. Two-tailed student t-test analysis was performed between knockouts and respective controls. No statistically significant differences were found. (TIF)AcknowledgmentsWe would like to thank the IMM animal facility and flow cytometry units for technical assistance and Dr. Frank Costantini for RetMEN2B mice.Author ContributionsConceived and designed the experiments: ARMA HV-F. Performed the experiments: ARMA SA-M DF-P HR HV-F. Analyzed the data: ARMA SA-M DF-P HR HV-F. Contributed reagents/materials/analysis tools: RL VP. Wrote the paper: ARMA HV-F.
Amino acid odorants are widely used olfactory stimuli for aquatic vertebrates like fish [1?], amphibia [5?], as well as aquatic invertebrates [8?0]. As protein decomposition, in particular food decomposition, generates amino acids, these stimuli have been proposed to serve as cues in the search for food [11?3]. Olfaction in vertebrates begins with the 23727046 binding of odorants to olfactory receptors (ORs) located on cilia or microvilli of olfactory receptor neurons (ORNs) situated in the olfactory epithelium (OE). The activation of ORs triggers the activation of G-proteins, which in turn initiate transduction cascades generally leading to depolarization of the ORNs and to receptor potentials (for a review see [14]). The ORs for amino acid detection are as yet, with few exceptions [15,16], unknown, and the concentrations of amino acids that have been used to stimulate individual ORNs were rather high in some physiological studies (e.g. [3,5,6,8,9,17,18]). Furthermore, it is known that protein decomposition also generates a considerable amount of soluble peptides [19]. Also, except for a study in the rainbow trout by Hara [20], and with the exception of peptide ligands of major histocompatibility complex (MHC) molecules [21,22], to the best of our knowledge peptide odorants have so far not been tested in aquatic species. One might thus question whether amino acids are the natural and adequate stimuli for the ORs they bind to. Alternatively, these receptors could be peptide receptors which also bind.D [18,36]. Hprt1 was used as housekeeping gene. For TaqMan assays (Applied Biosystems) RNA was retro-transcribed using High Capacity RNA-to-cDNA Kit (Applied Biosystems), followed by a pre-amplification PCR using TaqMan PreAmp Master Mix (Applied Biosystems). TaqRET Signalling and T Cell Developmentsequence. D. In order to evaluate the activity of Cre recombinase driven by hCD2, we bred hCD2Cre-expressing animals to Rosa26 eYFP animals. Histograms show flow cytometry analysis of eYFP expression in DN1 to DN4 thymocytes. (TIF)Figure S3 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. Results show absolute numbers of DN1 N4 (top) and DN to mature single positive (bottom) in hCD2Cre/Retnull/fl (open circle) and control hCD2Cre2/Retwt/fl (full circle) mice. Mean value: dash line. All WT and conditional Ret knockout deficient pairs were compared using two-tailed student t-tests, and no significant differences were found except where noted. *p,0.05. (TIF) Figure S4 Impact of Ret gain-of-function mutation RetMEN2B in adult thymic development. 8 week old RetMEN2B/MEN2B (MEN2B) and their WT littermate controls wereanalyzed by flow cytometry. Results show absolute numbers of DN1 N4 (top) and DN to mature SP (bottom) in MEN2B (open squares) and WT control (full circle) mice. Mean value: dash line. Two-tailed student t-test analysis was performed between knockouts and respective controls. No statistically significant differences were found. (TIF)AcknowledgmentsWe would like to thank the IMM animal facility and flow cytometry units for technical assistance and Dr. Frank Costantini for RetMEN2B mice.Author ContributionsConceived and designed the experiments: ARMA HV-F. Performed the experiments: ARMA SA-M DF-P HR HV-F. Analyzed the data: ARMA SA-M DF-P HR HV-F. Contributed reagents/materials/analysis tools: RL VP. Wrote the paper: ARMA HV-F.
Amino acid odorants are widely used olfactory stimuli for aquatic vertebrates like fish [1?], amphibia [5?], as well as aquatic invertebrates [8?0]. As protein decomposition, in particular food decomposition, generates amino acids, these stimuli have been proposed to serve as cues in the search for food [11?3]. Olfaction in vertebrates begins with the 23727046 binding of odorants to olfactory receptors (ORs) located on cilia or microvilli of olfactory receptor neurons (ORNs) situated in the olfactory epithelium (OE). The activation of ORs triggers the activation of G-proteins, which in turn initiate transduction cascades generally leading to depolarization of the ORNs and to receptor potentials (for a review see [14]). The ORs for amino acid detection 
are as yet, with few exceptions [15,16], unknown, and the concentrations of amino acids that have been used to stimulate individual ORNs were rather high in some physiological studies (e.g. [3,5,6,8,9,17,18]). Furthermore, it is known that protein decomposition also generates a considerable amount of soluble peptides [19]. Also, except for a study in the rainbow trout by Hara [20], and with the exception of peptide ligands of major histocompatibility complex (MHC) molecules [21,22], to the best of our knowledge peptide odorants have so far not been tested in aquatic species. One might thus question whether amino acids are the natural and adequate stimuli for the ORs they bind to. Alternatively, these receptors could be peptide receptors which also bind.
