Figure 1. Mapping and lineage tracing of pulmonary neuroendocrine cells.

(A-E) Confocal images of NE cells in bronchial epithelium of embryonic day (E) 18 (A-D) or adult (postnatal 2 month; E) Ascl1^CreER/+;Rosa26^ZsGreen/+mouse induced with tamoxifen at E14 to label NE cells with ZsGreen (green). See also Figure S1. (A,B) Solitary NE cells with pyramidal (A) and slender (B) morphologies. Arrowhead, thin projection to lumen. (C) Mini-cluster of 4 NE cells. (D) Neuroepithelial body (NEB), a large cluster of ~25 NE cells (12 visible in optical plane). (E) Adult NEB. Cells are more uniformly columnar. Note non-NE cells (nuclei marked by dots) superficial to NE cells in NEBs (D,E). Sample was also stained with PGP9.5 (red) to confirm NE identity and show innervating fibers (arrowhead). Dotted lines, individual NE cells in clusters; dashed lines, basement membrane location determined by co-staining with E-cadherin or laminin γ1. Bar, 10 μm.

(F-I) Stereotyped locations of NEBs at airway branchpoints. (F) Left primary bronchus (L) and some daughter (L.L1, L.L2, ...) and granddaughter (L.L1.A1, L.L1.A2,...) branches showing positions of NEBs at E18. (G) Low magnification confocal image of left primary bronchus of E18 Ascl1^CreER/+;Rosa26^ZsGreen/+ mouse. Note pairs of NEBs (green) at indicated branch points (L.L2, L.M3), with one NEB located proximal (p) and the other distal (d) along bronchial tree. (H) Longitudinal optical section through base of L.L2 showing L.L2p and L.L2d NEBs. Closeups of NEBs (boxed) are shown at right. Bar, 5 μm. (I) Schematic longitudinal section (LS) and cross section (XS) showing diametrically-opposite positions of L.L2 NEBs at branch base. (See also Figure S2).

(J-M) Lineage tracing of NE cell origins. Confocal images of NEBs (J,L) and solitary NE cells (K,M) at E18 from Shh^Cre/+;Rosa26^mTmG/+ mouse to label bronchial epithelial lineage (J,K) or from Wnt1-Cre;Rosa26^{ZsGreen /+} mouse to label neural crest lineage (L,M). Lungs were co-stained for lineage tag (green) and NE marker CGRP (magenta). All NEB and solitary NE cells express bronchial trace and none express neural crest trace. Other epithelial cells also express bronchial trace, and sub-epithelial neurons (arrowheads) express Wnt1 trace. Dots, outline of NEBs or individual NE cells; dashes, basement membrane. Bar, 10 μm.