(a) Illustration of cell fate-tracing strategies. Neonatal mice were injected either at P0 with EdU, or at P3 with Tamoxifen (TAM). The efficiency of recombination of RetCreERT2;R26RTOMATO and TrkACreERT2;R26RTOMATO mouse lines was examined at P5 and analyses were performed in animals sacrificed at P11. (b) Triple immunohistochemistry for NPY, EdU and RET on SG sections from P11 wild-type mice injected with EdU at P0. RET+NPY+ neurons (inset) were rarely EdU+ (arrowheads). (c-f) Recombination efficiency and validation of the RetCreERT2;R26RTOMATO mouse strain for tracing of cholinergic neurons. (c) Double immunohistochemistry for PROX1 and GFP on SG sections from P3 RetCFP mice. PROX1+ cells expressed low levels of RET (RetlowPROX1+ cells, arrow), when compared to cholinergic RethighPROX1- cells (arrowhead). (d) Double immunohistochemistry for TOMATO (recapitulating RetTOM) and cholinergic markers VIP or TLX3, on SG sections from P5 RetCreERT2;R26RTOMATO mice injected with Tamoxifen at P3, showing TOMATO+ cells expressing markers of the cholinergic lineage of sympathetic neurons. (e) Triple immunohistochemistry for TOMATO (recapitulating RetTOM), PROX1 and VACHT on SG sections from P5 RetCreERT2;R26RTOMATO mice, injected with Tamoxifen at P3. The vast majority of RetTOM+ cells at P5 were VACHT+ showing that this line can be used to trace cells of a cholinergic origin. In rare cases, recombination occurred in PROX1+VACHT- cells expressing low levels of Ret (insets). (f) Quantification of (e). n = 2, data are presented as mean ± s.e.m.. Percentages refer to the proportion of each cell type within the total of RetTOM+ neurons. (g) Generation of a TrkACreERT2 mouse strain. The black boxes represent the exons of the Ntrk1 (TrkA) gene. An internal ribosomal entry site (IRES) directly followed by CreERT2 was introduced after the stop codon of the TrkA gene. The neomycin selection cassette was eliminated by Flp recombination to generate the TrkAIRES‑CreERT2 mouse line (abbreviated TrkACreERT2 line). (h-j) Recombination specificity of TrkACreERT2;R26RTOMATO mice. (h) Double immunohistochemistry for TOMATO (recapitulating TrkATOM) and PROX1 or VACHT, VIP, TLX3, confirms that TrkATOM cells are neither dividing cells nor cholinergic neurons. (i) All TrkATOM cells at P5 are TRKA+ and, very rarely, RET+ (arrowheads). (j) Quantification of (i). n = 3 per stage, data are presented as mean ± s.e.m.. Percentages in (j) refer to the proportion of each cell type within the total number of TrkATOM+ neurons. (b-d) and (h) are representative images from experiments carried out on at least two animals. Scale bars: 10 μm (c), (d), (i), (j); 20 μm (b), (e), (f).