Figure 7.

Recovery of Mash-1 expression by switching of lung explants from hypoxia to normoxia (reoxygenation) in short-term (1 day, a–d) and long-term (6 day, e–h) cultures. (a) Positive immunoreactivity for PGP9.5 (green signal) and Mash-1 (red signal) in NEB cells (arrow) from E13 lung in normoxia culture for 1 day. (b) Developmental onset of a more mature PNEC/NEB marker CGRP (green signal, arrow), expressed along with Mash-1 (red signal) by day 2 in normoxia culture. (c) In contrast, in parallel organ cultures exposed to 1 day of hypoxia, there is loss of Mash-1 and PGP9.5 expression from NEB cells (arrow), whereas PGP9.5 immunoreactivity in adjacent ganglion cells is preserved (double arrows). (d) When hypoxia cultures are switched back for 1 day of normoxia, there is a robust recovery of Mash-1 expression (arrow) in NEB cell nuclei (red signal); however, note the absence of CGRP expression as compared with (b) (original magnification × 630). Long-term organ cultures initiated at E12 showed a strong expression of both PGP9.5 and Mash-1 in NEB cells (arrow) in cultures maintained in normoxia for 6 (e) or 7 days (f), whereas in organ cultures exposed to hypoxia (g) over the same time period, only very few Mash-1- and no PGP9.5-positive cells are evident. Double arrows indicate positive PGP9.5 staining in ganglia. (h) When these cultures are then switched back to normoxia for 1 day, there is a robust recovery of Mash-1 and PGP9.5 expression in NEB cells (arrow). Parallel control organ cultures in normoxia (f) maintain PGP9.5 and Mash-1 expression in NEB cells (arrow) (original magnification × 400).