Fig. 1: Cholinergic expansion during haematopoietic regeneration.

a, c Acetylcholine (ACh) content (a; N = 5,9,14; P = 0.03) and Gfra2 mRNA expression (c; N = 5,4,7; P = 0.002,0.0008) in endosteal BM before or 2–4 weeks after transplantation. b, d Density of (b) cholinergic (GFRα2⁺; N = 4,5,9; P = 0.003,0.02) or (d) noradrenergic (tyrosine hydroxylase, TH⁺; N = 4,8,6; P = 0.01) genetically marked nerve fibres in Wnt1-Cre2;Ai14D mice. e, f Representative genetic tracing showing the expansion of cholinergic stromal cells (red) adjacent to Nes-GFP⁺ BMSCs (green) associated with CD31⁺ or endomucin (EMCN)⁺ blood vessels (white) near the growth plate of ChAT-IRES-Cre;Ai14D;Nes-GFP tibias (N = 3). Nuclei are counterstained with DAPI (blue). Scale, 50 μm. g, h Transient expansion of endosteal BM cholinergic PDGFRα⁺Sca1⁻ skeletal stem cells (SSC), PDGFRα⁺Sca1⁺ (PαS) cells, PDGFRα⁻CD51⁺Sca1⁺ bone-lining osteoprogenitors (OPCs) or PDGFRα⁻CD51⁺Sca1⁻ osteoblast precursors (OBPs) genetically traced in (g) ChAT-Gfp mice (0 weeks, SSC, PαS, N = 9. OPC, OBP, N = 4; 2 weeks, SSC, PαS, N = 8. OPC, N = 7, OBP, N = 8; 4 weeks, SSC, PαS, OPC, OBP, N = 8) or (h) ChAT-Ires-Cre mice (0 and 4 weeks, N = 4; 2 weeks, N = 5). a, c, g, h Each dot is a mouse. Data are mean of biological replicates ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ANOVA and Tukey’s multiple comparisons test.