Sympathetic cholinergic signals locally repress adhesion to BM vessels during the daytime. Z-stack projection showing immunofluorescence of CD31⁺ endothelial cells (blue) and VAChT⁺ nerve fibers (red) in the skull of WT (A) and Gfra2^−/− (B) mice. Scale bar, 100 μm. (C) Quantification of VAChT⁺ fibers in the skull periosteum of Gfra2^−/− and WT mice. Immunofluorescence of CD31⁺ endothelial cells (blue) and Gfrα2⁺ nerve fibers (red) in the skull of WT (D) and Nrtn^−/− (E) mice. Scale bar, 100 μm. (F) Quantification of Gfrα2⁺ fibers in the skull periosteum of Nrtn^−/− and WT mice. Gfra2 (G) and Nrtn (H) mRNA expression at ZT13 in the BM of Gfra2^−/−, Nrtn^−/−, and WT mice. (I) Normalized WBC counts in peripheral blood of Gfra2^−/−, Nrtn^−/−, and WT mice at ZT13. Vcam1 (J) and Sele (K) mRNA expression at ZT13 in the unfractionated BM of control Nrtn^+/+ and compound Nrtn^−/− mice. (L) Frequencies of donor-derived WT lin⁻sca1⁺ckit⁺ HSPCs that homed to the BM at ZT5, 6 hours after IV transplantation into nonirradiated Gfra2^−/−, Nrtn^−/−, and WT mice (at ZT23). (M) Blood CFU-C fold change at ZT5 in WT mice treated with saline, β-AR antagonists, or cholinergic nicotinic (Chrn) antagonist at ZT23. Vcam1 (N) and Sele (O) mRNA expression at ZT5 in the BM of WT mice treated with saline, β-AR antagonists, or cholinergic nicotinic antagonist at ZT23. (C,F-O) Data are mean ± standard error of the mean; n (inside bars) and P values (multivariate analysis for >2 groups) are indicated. *P < .05, **P < .01, ***P < .001, unpaired 2-tailed t test (C,F,J-K), 1-way analysis of variance with Bonferroni comparisons (G-I,L-O). ns, not significant.