Fig 2: Multiple doses of CV maintains physiological saliva secretion, acinar cell architecture, and functional innervation in the long term.(A) Timeline for radiation, treatments, and saliva collections. (B) Citrate-induced saliva output was measured once every 2 weeks from 35 to 91 days after radiation. (C) Maximal saliva secretion in response to systemic pilocarpine administration at 92 days. Data were normalized to non-IR controls. (D to G) Representative images and quantification of AQP5+ (D and E) and MUC10+ (F and G) acinar cells. Epithelial cells and nuclei were stained for E-cadherin (CDH1, red) and nuclei (Hoechst 33342, blue). (H to K) Representative images (H) and quantification of mitotic (pHH3+) acinar progenitor cells (I) (SOX2+) and transit-amplifying cells (J) (SOX2-). Quantification of SOX2+ acinar cells is shown in (K). (L to N) Representative images (L) and quantification of parasympathetic nerves marked by GFRa2 (M) [green, three-dimensional (3D) surface reconstructed with Imaris] and the neuropeptide VIP (N) (red, 3D surface reconstructed using Imaris). Hoechst 33342, nuclei (blue). GFRa2+ filaments with diameters of less than 1.5 µm (innervating the acini) were quantified. VIP was normalized to GFRa2+ filament volumes. (O to Q) Representative images (O) and quantification of pro-reparative (CD68+CD206+) M2-polarizing macrophages (P) and CD3+ T cells (Q). Scale bars, 40 µm (D, F, H, and O) and 10 µm (L). Dots in the bar graphs represent biological replicates. Data are expressed as means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; a one-way ANOVA with Dunnett’s multiple comparison test for all graphs except in (B); a two-way ANOVA with Dunnett’s multiple comparison test was applied to (B).

Fig 3: Acinar cells undergo a time-limited regenerative response 1 week following radiation exposure that correlates with increased neuronal activity.(A) Heatmap showing mitotic, immune, and neuronal gene expression in sublingual glands at 7 or 14 days post-IR, compared to non-IR controls, n = 3 mice per group. (B) GO analysis of significantly up-regulated genes (fold change > 1.5, P < 0.05). (C) Sublingual glands from non-IR and 7- and 14-day post-IR mice immunostained for pHH3 (red), NKCC1 (green), and nuclei (blue). Arrows highlight proliferating acinar cells. pHH3+ acinar cells were quantified and expressed as a percentage of the total acinar cells (graph). (D to F) Immunofluorescent detection of EdU+ cells (green) in sublingual glands at 2 hours and 7 days following a single injection of EdU into non-IR mice (D) and mice at 7 days (E) and 14 days (F) post-IR. Arrows highlight proliferating acinar cells. EdU+AQP5+ cells were quantified and expressed as a percentage of the total acinar cells (graphs). (G) Citrate-induced saliva collection at 7 and 14 days post-IR and in non-IR control. (H and I) Quantification (H) of parasympathetic nerves (GFRa2, red) innervating acini (AQP5, green) in sublingual glands at 14 days post-IR versus non-IR control. Nuclei, blue. Scale bars, 40 µm (C to F) and 15 µm (I). Each dot in the bar graph represents a biological replicate. Data are expressed as means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; graphs (D) to (F) and H): an unpaired two-tailed t test for two-group comparisons. Graphs (C) and (G): a one-way ANOVA followed by a Dunnett’s test for multiple comparisons.

Fig 4: Reduced HSC self-renewal in a cholinergic-neural-deficient niche.a Scheme illustrating competitive serial HSC transplantations. 500 endosteal LSK CD48- CD150+ HSCs isolated from primary recipients were i.v. injected together with 105 BM helper cells into lethally irradiated secondary recipients of the same genotype as the primary recipient mice. b–g Donor-derived haematopoietic chimerism (b), B cells (c), T cells (d), monocytes (e), eosinophils (f) and neutrophils (g) and at specified time following secondary transplantation of mice with specified genotype (Gfra2+/- > CD45.1, N = 3; Gfra2-/- > CD45.1, N = 4; CD45.1 > Gfra2-/-, N = 4). Data are mean of biological replicates ± SEM. *P < 0.05, ***P < 0.001. ANOVA and Tukey’s multiple comparisons test.

Fig 5: Reduced quiescence and self-renewing programme in endosteal HSCs from cholinergic-neural-deficient mice.Gene sets enrichment analysis (GSEA) comparing endosteal and central lin-CD48-sca1+ckit+CD150+ HSCs of WT mice and Gfra2-/- mice (N = 4 biological replicates). a Enrichment map showing upregulated pathways in central (compared with endosteal) BM HSCs. b Downregulation of RET target genes in HSCs from Gfra2-/- mice. c Enriched Notch1-Myc target gene expression in endosteal WT HSCs. d Datasets from the Reactome, Pathway Interaction Database and Hallmark database as well as published HSC datasets were used for comparison. The normalised enrichment score (NES) of significantly enriched datasets (False discovery rate (FDR) < 0.1) is depicted as heatmap. Red, blue, and white colours indicate upregulation, downregulation, and no change, respectively.