Fig 1: Characterization of the cholinergic system in bone(A) Immunofluorescence of pan-neural TUJ1 in Nes-GFP femur. Insets show bone (A') and growth plate (A") areas. Scale bars, 500 µm (A) and 100 µm (A' and A").(B and C) Immunofluorescence of osteolineage markers in WT long bones with high-magnification insets.(D) Frequency of TUJ1+ cells among Nestin-GFP+ and osteolineage cells expressing RUNX2 or osteolectin.(E and F) Genetic tracing of cholinergic cells in (E) cortical bone and (F) growth plate of ChAT-IRES-cre;Ai35D mice. Scale bars, 200 µm. See also Figure S1C.(G and H) Immunofluorescence of VAChT+ cholinergic nerve fibers in (G) periosteum and (H) cortical bone in WT femur. See also Figures S1E–S1G.(I and J) Immunofluorescence of VIP+ or VAChT+ cholinergic nerve fibers in cortical bone of (I) a Nes-GFP mouse, or (J) WT or GFRa2 KO mice.(K and L) Area covered by (K) VAChT+ or VIP+ cholinergic nerve fibers in cortical bone or (L) VAChT+ cells near the growth plate of WT or GFRa2 KO mice.(M) Immunofluorescence of VAChT+ cells in the growth plate of WT or GFRa2 KO tibias. Scale bars, 200 µm.(N) Summary of changes in pan-neural and cholinergic markers in GFRa2 KO mice. N.A., not assessed.(B, C, and G–J) Scale bars, 100 µm.(D, K, and L) Data are mean ± SEM; **p < 0.01, unpaired two-tailed t test.(A–I) Arrowheads depict nerve fiber staining and arrows depict non-neural staining.(A–J and M) Nuclei were counterstained with DAPI (blue). EMCN, endomucin.