Fig 3. tGFP (GFP) expression by major lung cell subtypes.

A) The definitive Club cell marker, CC10, is shown to co-localize with GFP (arrows). Other GFP staining in the bronchial lining includes neuronal axons (arrow head; see Fig 2). Bar = 15 microns. B) Examination of Club cell anti-CC10 staining revealed by a peroxidase stained section of both α7^G and α7^E260A:G distal bronchial sections is shown at two magnifications. The panels to the right show greater magnification. In the lower magnification images (panels to the left) the typical appearance of the relatively smooth surface of the α7^G bronchia and including evenly distributed Club cells (arrows) is observed. In contrast, the α7^E260A:G bronchial lining is more uneven and Club cells appear to protruded from the bronchial surface (arrows). In the α7^E260A:G lung this includes aggregate-like clusters (arrowheads) of these cells that are rare or absent from the α7^G lung. This is particularly evident in the images of increased magnification to the right. The CC10-labeled Club cells (arrows) are evident and again in the α7^E260A:G commonly protrude into the airway space. Relatively large aggregates (arrow heads) are also evident. Bars = 25 microns (left panels) and (right panels) = 15 microns. C) Merged images of immune-localization of GFP (green) and acylated-α-tubulin (acTub, red) shows ciliated cells (arrows) typical of α7^G (left panel) or in the α7^E260A (right panel) lung. No co-expression of tGFP and acTub was observed. Also the ciliated cells of the α7^E260A mouse appeared to be fewer and of altered morphology relative to the control. Bar = 15 microns. D) Sections similar to those from (C) but stained using peroxide to reveal acTub in ciliated cells. Consistent with fluorescent immunostaining, ciliated cells are relatively evenly dispersed in the α7^G lung bronchial linings and prominent well-defined cilium are present. In the α7^E260A:G lung similar staining shows ciliated cells that tend to be dispersed further apart that often lack well defined cilia (arrows). E) A longitudinal section reveals the bronchial surface facing the airway and the distribution of acTub stained ciliated cells (arrows). Their distribution and the cilium complexity differ between these α7-genotypes and the regular networks formed by ciliated cells (asterisk) in the α7^G lung are absent in the α7^E260A:G. Bar = 15 microns. F) Quantitation of ciliated cell number comparing the α7^G to the α7^E260A:G mouse. The average percent of ciliated cells per unit measure was made from 5 sections from each of 3 mice of the identified α7 genotype. A highly significant difference shows that α7^E260A:G lung harbors approximately one-third fewer ciliated cells relative to the α7^G lung. G) Co-expression of GFP and the ATII cell marker, surfactant protein c (Sftpc; arrows). The arrow head identifies an AM. H) The GFP staining is compared to aquaporin 5 (Aqp5), a marker of ATI cells (asterisk). Essentially no overlap in expression between these markers was observed. An ATII cell is identified by an arrow and an AM by an arrowhead. Staining for these cells was essentially identical between α7^G (shown) and α7^E260A:G mice with differences to be discussed. For G and H the Bars = 15 microns.