Figure 4.

Parasympathetic deficiency increases nocturnal HSPC BM adhesion and homing through β₂-adrenergic signaling in the microenvironment. Vcam1 (A), Sele (B), and Selp (C) mRNA expression at ZT13 in the unfractionated BM of control Gfra2^+/− mice, Gfra2^−/− mice, single β₂-AR (Adrb2)-deficient or β₃-AR (Adrb3)-deficient mice, or compound Gfra2^−/−Adrb2^−/− and Gfra2^−/−Adrb3^−/− mice. Vcam1 (D), Sele (E), and Selp (F) mRNA expression at ZT13 in CD45⁻Ter119⁻ endothelial (CD31⁺) or nonendothelial (CD31⁻) cells from Gfra2^−/− and control Gfra2^+/− mice. (G) Scheme showing the protocol used for blockade of in vivo HSPC adhesion to blood vessels using antibodies against α₄-integrin, P-selectin, and E-selectin (IV injection at ZT11 and analysis at ZT13). (H) HSPCs circulating at ZT13, 2 hours after injection of blocking antibodies (Abs) or control IgG. Please note that CFU-C fold change goes from a 3.2-fold increase in control immunoglobulin G–treated mice to a 1.3-fold increase in blocking antibody–treated mice. (I) Frequencies of donor-derived WT CD45.1⁺ lin⁻sca1⁺ckit⁺ HSPCs at ZT2 that homed during the night to the BM after IV transplantation (at ZT10) into nonirradiated Gfra2^−/− mice or control Gfra2^+/− mice preconditioned with saline or β₂ adrenergic antagonist (ICI118,551) 4 hours before transplantation. (A-F,H-I) 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, 1-way analysis of variance with Bonferroni comparisons. ns, not significant.