Mouse body weights.

<p>*p<0.0001 vs <i>Sema3A^+/+</i>.</p><p>**p<0.0001 vs <i>Sema3A^+/-</i>.</p

Sema3A expression.

<p><b>A.</b> Reduced Sema3A expression in the lungs of <i>Sema3A^-/-</i> mice was assessed using qPCR. <b>B.</b> Representative AP-Nrp-1^ecto binding in tissue sections from <i>Sema3A^+/+</i> (middle panels) and <i>Sema3A^-/-</i> mice (bottom panels) at P1 (left) and P28 (right). Prominent AP-Nrp-1^ecto binding was seen in bronchiolar epithelium of littermate control (<i>Sema3A^+/+</i>) mice at both P1 and P28. This pattern of tissue binding of AP-Nrp1^ecto was absent in lung sections from <i>Sema3A^-/-</i> animals, suggesting specificity of the signal for Sema3A expression. Top panels represent non-specific AP staining in lung sections hybridized with a control AP-vector. (Magnification  =  20x).</p

ATI markers are reduced in <i>Sema3A^-/-</i> mice.

<p><b>A.</b> Reduced immunostaining for the type I alveolar epithelial cell marker, T1-alpha (red), is seen in lung from a <i>Sema3A^-/-</i> mouse at E17.5 (right) as compared with a <i>Sema3A^+/+</i> littermate control (left). Nuclei are counterstained with Fast Yellow (green). Results are representative of 3 animals/group. (Magnification = 10x). <b>B.</b> Western blotting demonstrates decreased expression of both T1-alpha (left) and Aqp5 (right) protein in lung homogenate from <i>Sema3A^-/-</i> animals as compared with <i>Sema3A^+/+</i> littermate controls. Results are representative of 7-9 animals/genotype.</p

Lung morphology is abnormal in <i>Sema3A^-/-</i> mice.

<p><b>Top panel.</b> Representative lung histology from <i>Sema3A^-/-</i> mice (right panels) and <i>Sema3A^+/+</i> littermate controls (left panels) at E17.5, P1, P14 and P28. Prominent septation defects were seen in all <i>Sema3A^-/-</i> mice that survived to P14 or beyond. At the earlier ages, septae of lungs from many <i>Sema3A^-/-</i> mice were thickened, and clusters of cellular debris were sometimes detected in airspaces at P1 (black arrowheads in panel H). Scale bar represents 50 µm. <b>Bottom panel.</b> Airspace size was quantitated morphometrically by calculation of the mean linear intercept (MLI). MLI increased in all <i>Sema3A^-/-</i> mice (white bars) surviving to P14 or beyond as compared with <i>Sema3A^+/+</i> littermate control animals (black bars) and <i>Sema3A^+/-</i> littermates (see text). Data represent mean ± SE for 4 mice/group at each time point.</p

Lung cell death but nor proliferation is altered in <i>Sema3A^-/-</i> mice.

<p><b>A.</b> Widespread immunostaining for the proliferative marker PCNA (green) was seen in lungs from both E17.5 <i>Sema3A^+/+</i> and <i>Sema3A^-/-</i> mice (top panels). By P1 (middle panels) and P14 (lower panels), PCNA staining was largely confined to cells lining small airways, and did not differ between <i>Sema3A^-/-</i> animals and littermate controls. Nuclei are counterstained with DAPI (blue) (Magnification = 40x). <b>B.</b> Shown graphically, the average proliferative index did not significantly differ between <i>Sema3A^-/-</i> (white bars) and <i>Sema3A^+/+</i> (black bars) mice at any time point evaluated (bars represent mean ± SE for n = 3 animals/group). <b>C.</b> Representative TUNEL labeling (green) in lungs from <i>Sema3A^-/-</i> mice surviving to P1 (top right panel) and P14 (bottom right panel). Shown for comparison in the left panels are lungs from <i>Sema3A^+/+</i> littermate controls. Nuclei are counterstained with DAPI (blue). (Magnification  = 40x). <b>D.</b> Apoptotic index increased significantly in <i>Sema3A^-/-</i> animals (white bars) as compared with <i>Sema3A^+/+</i> littermate control mice (black bars) at both P1 and P14 (n.d. =  not detected; bars represent mean ± SE for n = 3 animals/group).</p

iNOS staining in airways of vagotomized mice.

<p><b>A.</b> Increased iNOS staining (brown) in bronchial epithelium of adult mouse treated with immunosuppressive agents (Black arrow). iNOS staining was qualitatively increased in the bronchial epithelium of five of the five drug-treated mice. <b>B.</b> Minimal iNOS staining in airway of adult mouse that was not treated with immunosuppressive agents. Minimal iNOS staining was observed in five of the five mice not treated with immunosuppressive agents. <b>C.</b> No first antibody control.</p

Representative examples of ciliated epithelial cells in the trachea of control and drug-treated mice.

<p>Tissue was evaluated at 100 X and at 40 X magnification for the control (A and B) and drug-treated mice (C and D), respectively. Black arrows point to cilia. Qualitatively, the presence of ciliated epithelial cells in the airways in each of the representive animals is similar.</p

PAS staining of seromucous cells in tracheal glands of vagotomized mice.

<p><b>A.</b> Trachea of mouse treated with immunosuppressive agents. <b>B.</b> Trachea of mouse not treated with immunosuppressive agents. Black arrows point to PAS staining in seromucous cells. Qualitatively, both the drug-treated animals and control mice appeared to have similar PAS staining in the seromucous cells of the tracheal glands.</p

Mean MCC (±SD) from the right lung between 1–1.5 hours and 6–6.5 hours in 7 C57BL/6 control mice (dark bar) and 8 drug-treated C57BL/6 mice (light bars).

<p>There was a trend toward slower clearance in the drug-treated mice, compared to controls, at 1–1.5 hours, but differences in MCC were not statistically significant. Mucociliary clearance was statistically significantly slower in the drug-treated mice, compared to controls, at 6–6.5 hours (p = 0.006).</p