Figure 4.

PGP9.5 Immunostaining in E11.5 lungs treated with npas1- or arnt-antisense. (a, b) Immunostaining of lung buds cultured for 6 d with npas1-sense oligos have occasional clusters of PGP9.5-positive NE cells scattered throughout the airway epithelium (long dark arrows) and also a few isolated NE cells (short thin arrows). As an internal positive control, a PGP9.5-positive nerve fiber is visible in the mid-left field of a (solid arrowhead). Bars in lower left hand corner = 50 μm. * Pleural surface. (c, d) Lungs cultured with npas1 AS for 6 d have a marked increase in PGP9.5-positive NE cell clusters (solid arrows), as well as a modest increase in isolated NE cells (thin arrows). Small linked arrows in lower right hand corner of c indicate a stretch of linear NE cell hyperplasia. The internal positive control is a PGP9.5-positive nerve fiber (arrowhead, upper left hand corner of c, in which asterisk indicates pleural surface). Bars in lower left hand corner = 100 μm (c) and 50 μm (d). (e) Morphometric analysis for relative numbers of PGP9.5+ NE cells in E11.5 lung buds treated with npas1-sense or -AS ODN in culture for 6 d. We determined the number of PGP+ cell foci (including both isolated cells and clusters of ⩾ 2 cells) and the total number of PGP+ cells, both of which were normalized for the area of lung tissue present using Scion Image 1.62 software (Scion Corp., Frederick, MD) and density slice analysis. The number of PGP+ cells per focus was also calculated. Data shown represent the mean values ± SE for complete cross-sections of n = 9 (sense-treated lung buds) and n = 14 (AS-treated lung buds), representing pooled data from four experiments including matched controls from the same litters. * P < 0.0001 compared with npas1 sense control; ^¥ P < 0.012 compared with sense control.