Fig. 1.

Elastin remodeling during lung development. A: quantitation of total elastin in situ zymography signal demonstrated increasing lung elastase activity starting on postnatal day (PND) 1 and increasing activity at PND7 and PND14 compared with earlier time points. Adult mouse lung had very little elastin remodeling. B: elastin remodeling localized to major airways and vessels (white bars) on PND1. Nonseptal tip elastin remodeling (gray bars) increased during late saccular and alveolar development (PND3-PND14), with relatively consistent septal tip elastin remodeling (black bars). For clarity, only statistical comparisons between nonseptal tip elastin are noted. C: normalizing elastase signal to tropoelastin signal demonstrated that increased nonseptal tip elastin remodeling is not due to increased tropoelastin content. D: in E15.5 lung sections, tropoelastin (red) was localized to the mesenchyme underneath the airway epithelium, and the small amount of elastase activity (green) present was located within the epithelial layer. DAPI staining is blue. Scale bar = 150 μm. E: in PND1, lung section elastin remodeling was most pronounced in major vessels (white arrows), with relatively less activity in major airways, although major airways accounted for a larger percentage of total lung volume. F: PND3 lung sections demonstrated continued arterial elastin remodeling and increased remodeling of airspace walls. G: at PND7, early in the alveolar stage of lung development, elastin remodeling in arteries and major airways was relatively diminished with increased elastase activity in airspace walls (*) and appreciable activity in septal tips (white arrow). H: this remodeling of alveolar walls was accentuated in PND14 lung. I: in the adult lung (PN 8 wk), little elastin remodeling was detected. All presented images were processed identically and obtained on the same day with the same microscope and laser settings. J: 3X3 stitched X20 confocal z-stacks stained as above were used to quantitate and localize elastase activity. Images from three mice from different litters provided the quantitative data used for analysis. Scale bar = 500 μm. K: masks were created to define elastase signal (green) with manual masks defining major airways and vessels (mustard-colored shapes on right half of image). L: the tropoelastin signal (red) was masked based on intensity and sphericity to define tropoelastin structures as elastin tips (magenta) or nontips (mustard color). * P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA.