Figure 3. Autonomic nerve activity regulates alveolar formation.

(A, B) H&E staining of lung sections from control and Sox10^Cre/+; Ptox/+ mice (n=6 pairs) at P5.

(C) Measurement of the MLI in control and Sox10^Cre/+; Ptox/+ lungs (n=3 pairs) at P0, P3 and P5.

(D, E) Immunofluorescence of lung sections from control and Sox10^Cre/+; Ptox/+ mice (n=3 pairs) stained with anti-SMA (smooth muscle actin), anti-CD31 and phalloidin at P5. CD31 labels endothelial cells and phalloidin binds to F-actin. Arrows point to secondary septation.

(F, G) Immunohistochemical analysis of control and Sox10^Cre/+; Ptox/+ mouse lungs at P3. Immunoreactivity of SYP, which marked nerve fibers, showed no apparent difference between control and mutant lungs.

(H, I) H&E staining of lung sections from control and Ptox-mutant mice (conditional induction by tamoxifen).

(J) Measurement of the MLI in control and Sox10^iCreER/+; Ptox/+ lungs (n=5 pairs) at P10. Sox10^iCreER/+; Ptox/+ mice were injected with tamoxifen at 15.5 dpc and lungs were collected at P10.

(K-M) H&E staining of lung sections from control, TrkA^f/f; Th^Cre/+ and Th^Cre/+; Ptox/+ mice at P28.

(N) Measurement of the MLI in control, TrkA^f/f; Th^Cre/+ and Th^Cre/+; Ptox/+ lungs at P28 (n=4 for each genotype).

(O-Q) Immunohistochemical analysis of control, TrkA^f/f; Th^Cre/+ and Th^Cre/+; Ptox/+ mouse lungs at P28. Immunoreactivity of TH, which marked sympathetic nerves, was reduced in TrkA^f/f; Th^Cre/+ lungs but not in Th^Cre/+; Ptox/+ lungs.

(R-T) H&E staining of lung sections from control and Chat^Cre/+; Ptox/+ mice at P14.

(U) Measurement of the MLI in control and Chat^Cre/+; Ptox/+ lungs at P14 (n=4 pairs).

Scale bars, 100 μm (A, B), 10 μm (D, E), 50 μm (F, G), 100 μm (H, I), 100 μm (K-M, R-T), 25 μm (O-Q). All values are mean ± SEM. (*) p<0.05; (**) p<0.01; ns, not significant (unpaired Student’s t-test and one-way ANOVA).