Figure 6.

Experimental lungs are developmentally arrested after transient Fgfr2b signaling inhibition at E14.5. (A) Experimental design: pregnant females carrying experimental and littermate control embryos were injected with a single Dox-IP at E14.5, and embryos were harvested at E18.5. (B) Brightfield images of the ventral side of the whole lung in control (a, b) and experimental (d, e) samples, as well as the lateral perspective of the accessory lobe (c and f). Apart from the drastic difference in overall lung size between experimental and control lungs (d vs. a), notice the complete failure of the experimental epithelium to form the numerous branches and alveoli seen in the control, as well as the reduced mesenchyme and thickness of the lobes seen in the experimental lungs (f vs. c). (meso. = mesothelium). Scale bar: (a, d) 1.5 mm, (b, e) 95 µm, (c, f) 375 µm. (C) Immunofluorescent images (a-t) for control and experimental lungs. Antibody staining for AT1 (Hopx) and AT2 (Sftpc) cells suggests an overall reduction in the number of these cells in experimental compared to control lungs (compare c and g to a and e). Furthermore, in the case of Hopx staining in the experimental lungs (see g and h), there are cells with a similar expression pattern to the AT1 cells seen in controls, but also epithelial cells displaying a sparse, much reduced expression pattern (compare cells in the structure labeled by asterisks in h vs. f). Staining for the proximal epithelial markers Scgb1a1 and acetylated alpha-Tubulin reveals ectopic and distal expression in experimental compared to control lungs (compare d and l to b and j). Finally, staining for Ki67 (m–p) and TUNEL (q–t) to assess proliferation and apoptosis, respectively, shows that, in general, there is little to no impact on proliferative processes in either the epithelial or mesenchymal compartments. (meso. = mesothelium, br. = bronchus). Scale bar: (a, c, e, g, i, k, m, o, q, s) 125 µm, (b, d, f, h, j, l, n, p, r, t) 25 µm.