Fig 2: Osteolineage cells contribute to the non-neuronal cholinergic system(A–E) Immunofluorescence of cholinergic bone-lining cells near the growth plate (gp) in (A–C) ChAT-IRES-cre;Ai35D and (D and E) ChAT-IRES-cre;Ai14D mice with co-staining of osteolineage markers. Arrows depict co-localization in high-magnification insets (B'–E"). 2HG, 2nd harmonic generation imaging of collagen. See also Figures S4A and S4B.(F) Quantification of co-localization of ChAT-labeled cells.(G and H) Immunofluorescence of cholinergic bone-lining cells near the (G) growth plate and (H) endosteal regions of ChAT-IRES-cre;Ai14D;Nes-GFP tibias. Arrows depict nerve fibers. Arrowheads depict osteolineage cells. See also Figures S4C and S4D.(A–E) Scale bars, 100 µm (A and B) and 200 µm (G and H).(I) Flow cytometry gating strategy for analysis of CD31-CD45-Ter119-PDGFRa+Sca1- (PaS-) cells, CD31-CD45-Ter119-PDGFRa+Sca1+ (PaS+) cells, CD31-CD45-Ter119-PDGFRa-CD51+Sca1+ (OPS+) cells, and CD31-CD45-Ter119-PDGFRa-CD51+Sca1- (OPS-) cells.(J) Frequency of ChAT-IRES-cre-traced osteolineage cells in endosteal or central BM. See also Figures S4E and S4F.(K) Acetylcholine content in osteolineage cells from WT and Nes-GFP mice.(L) Acetylcholine content in primary WT osteoblasts (OB) and osteocytes (OC) from digested WT bone fragments.(M) Schematic of CD51+ osteolineage cell isolation and culture.(N and O) Acetylcholine content (N) and qRT-PCR analysis (O) of cultured CD51+ osteolineage cells.(P) Acetylcholine content from endosteal and central BM serum of mice lacking an a7 nicotinic receptor in LepR-cre targeted niche cells 1 month after bone marrow transplantation.(F and J–P) Data are mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; unpaired two-tailed t test (F and J–O) or ANOVA and pairwise comparisons (P).

Fig 3: Irradiation increases stromal VEGF production, stromal-vascular BM niche hyper-activation, and ROS levels in adult bone(A–C) ROS analysis in femurs of irradiated mice (n = 3), in (A) non-hematopoietic cells (CD45-Ter119-), (B) ECs (CD45-Ter119-CD31+), and (C) OPCs (TN CD51+). MFI, mean fluorescence intensity, relative to non-irradiated mice (n = 4).(D and E) Emcn IHC on tibias of irradiated (n = 3) versus non-irradiated mice (n = 4).(F–H) Flow cytometry for the (F) CD31+ EC fraction among non-hematopoietic BM cells, (G) Sca-1+ and Sca-1- EC fractions, and (H) absolute CD31+Sca-1+ cell numbers.(I and J) Flow cytometry for osteogenic stromal populations after irradiation.(K) VEGF qRT-PCR in femurs from irradiated mice (n = 2–5; one-way ANOVA with multiple comparison against the non-irradiated group).(L) VEGF qRT-PCR in BMSCs exposed to 7 Gy (n = 4 cell pools, each split into a non-irradiated and irradiated batch; paired t test).Graphs, mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, by (except in K and L) unpaired t test.

Fig 4: Responses of the stromal-vascular BM compartment to increased VEGF(A) Flow cytometry analysis of ROS levels by conversion of the probe dichlorodihydrofluorescein diacetate (DCFDA) to the highly fluorescent 2',7'-di-chlorofluorescein (DCF), in (left) ECs (CD45-Ter119-CD31+) and (right) hematopoietic cells (CD45+ and/or Ter119+) from E16.5 bones (n = 5–7).(B) DCF levels representing ROS in ECs of (left) arteriolar (CD45-Ter119-CD31+Sca-1+) and (right) sinusoidal (CD45-Ter119-CD31+Sca-1-) subtypes, derived from P2 bones (n = 5–7).(C) MIP (100-µm z axis) of CD31 and Emcn signals in E18.5 tibia BM cavities (dotted lines). Left, overviews; right, metaphyseal (top) and diaphyseal (bottom) regions.(D) Directionality of blood vessels quantified from segmented and skeletonized CD31 staining. Angle 0° = vessel running along the longitudinal bone axis; 90° = parallel to the growth plate zones. Right, area under the curve (AUC) for vessels with directionality between -45° and 45° (n = 4).(E and F) Number of blood vessels per mm2 (E) and distribution of vessels according to width (F), quantified on segmented CD31 staining images (n = 4).(G) MIP (60-µm depth) of CD31 and Emcn stained tibias at P2.(H) EC counts by flow cytometry (n = 4–8).(I) IHC for phospho-histone H3 (PHH3) and Emcn at P2, and Emcn+PHH3+ mitotic ECs quantified in the dashed areas of the metaphysis and diaphysis (n = 3).(J) Proportions sinusoidal (CD31+Sca-1-) and arteriolar (CD31+Sca-1+) ECs (n = 4–8).(K) MIP spanning 100-µm tissue depth of CD31, Emcn, and Osx-Cre:GFP signals on E18.5 tibias. Also see Video S1.(L) Nearest-neighbor distance analysis showing for distinct Osx-Cre:GFP+ cells the distance to the closest CD31+ or Emcn+ signal. Plots display individual values, median, and interquartile range (no statistics applied).(M) Proportion of triple-negative (TN; CD45-Ter119-CD31-) CD51+Sca-1- and CD51+Sca-1+ osteolineage progenitors in E18.5 limbs (n = 4–5).(N) TN PaS (PDGFRa+ Sca-1+) and PDGFRß+ cell numbers in (left) E18.5 and (right) P2 bones (n = 4–7).(O) MIP (20-µm depth) of PDGFRß, Emcn, and Hoechst signals in (top) E18.5 and (bottom) P2 tibias.(P) TEM of E16.5 tibias. Dotted lines, blood vessels; asterisks, lumens; orange arrows, irregular ruffled membranes; blue arrowheads, pinocytotic vesicles; green block arrows, mitochondria; red arrowheads, fenestrations/pores between zones of tight junctions (#).Graphs, mean ± SEM, except in (L). *p < 0.05, **p < 0.01, ***p < 0.001, t test. MFI, mean fluorescence intensity. See also Figures S6 and S7 and Video S1.