Fig. 5. Cell death of sympathetic neurons increases perinatally in Ret^-/- mice, owing to lack of non-GFL neurotrophic factors. (A-F) Thionin (A,B), Phox2b and BrdU (C,D) and activated-caspase 3 (E,F) staining of E16.5 STG. The STG of Ret^-/- embryos is mostly composed of neurons containing little cytoplasm (B), and with mixture of excessive dividing neuronal precursors (D) and dying cells (F). (G) Neuronal cell death is increased during the perinatal period in the SCG and STG of Ret^-/- animals. Error bars indicate the standard deviation. (H-M) Ret gene expression monitored by GFP immunohistochemistry in the SCG of Ret^TGM/+ and Ret^TGM/TGM animals at E11.5 (H,I), E15.5 (J,K) and P0 (L,M). Ret expression is markedly downregulated in most developing neurons in the SCG by E15.5. The Ret-expressing population in the SCG is comparable in size between Ret^TGM/+ and Ret^TGM/TGM mice at P0. The broken line demarcates the SCG (L,M). (N-Q) GFP (green) and activated caspase 3 staining (red) of the SCG of newborn Ret^TGM/+ (N, P) and Ret^TGM/TGM (O,Q) mice. Arrows (N,O) depict cells that are doubly stained. Despite the apparent increase of caspase 3-positive cells in Ret^TGM/TGM mice, GFP and caspase 3-positive populations remain largely distinct (P,Q). Scale bar: 35 µm in A-F, 140 µm in H,I,L,M,P,Q; 80 µm in J,K; 50 µm in N,O; 100 µm in P,Q.

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