Figure 3.

PNEC innervation during postnatal development was dependent on NT4. A) Quantification of solitary PNECs, clusters, and total number of PNECs at birth and at P7, 14, and 21 in WT mice. PNECs were quantified after CGRP staining of tissue sections collected from entire right cranial lobe. B) PNEC expression of NT4 by double staining for NT4 and CGRP. NT4^−/− lung sections were negative control for NT4 antibody. Arrows indicate PNECs. Scale bars, 100 µm. C) Representative images of postnatal TrkB lineage tracing using TrkB^CreERT2/+;Rosa(tmRed) mouse line. PNECs (arrows) were stained for CGRP indicated by arrows. Nerves (arrowheads) were labeled by tomato red (tmRed) fluorescence. Scale bar, 100 µm. D) Assessment of solitary PNECs and clusters in WT and NT4^−/− lungs with and without OVA challenges at P21. PNECs were labeled by CGRP staining of medial lung slices from left lobe. Each mark indicates results from 1 lung slice; n = 3 for each group. E) Assessment of NEB innervation in WT and NT4^−/− mice with and without OVA exposure. Lung slices were stained with CGRP and TuJ1 antibodies followed by confocal microscopy to assess spatial association between NEBs and nerves. NEBs with 5 or more PNECs were counted. NEBs without associated nerves (none), nerves at base (basal), and nerves penetrating into clusters (penetrating) were grouped and presented as percentages of all NEBs. F) Representative images of NEBs with penetrating nerves in WT and NT4^−/− mice with and without OVA challenges at P21. Scale bar, 50 µm. G) Quantification of NEB innervation density. Density of nerves was calculated by normalizing TuJ1-immunoreactive area by number of cells within NEBs in WT and NT4^−/− mice with and without OVA challenges. For data in E–G, total of 45–50 NEBs in lung slices from 3 mice were scored for each group. N.s., not significant. *P < 0.05.