Figure 4. Dlg5 is required for lung alveolar differentiation program.

(A–A′) Normal epithelial differentiation in E14.5 Dlg5^−/− lungs. Immunofluorescent stainings of lung sections from wild-type (Ctrl) and Dlg5^−/− embryos with anti-Sox2 antibodies.

(B–B′) Vasculature development in Dlg5^−/− lungs. Immunofluorescent stainings of E15.5 lung sections from wild-type (Ctrl) and Dlg5^−/− embryos with anti-PECAM (endothelial cell marker) antibody.

(C–E′) Immunofluorescent stainings of lung sections from E15.5 wild-type (Ctrl) and Dlg5^−/− embryos with anti-T1α and anti-pro-SPC (both expressed in epithelial lung cells at E15.5) antibodies. Boxed areas in C–C′ are shown with split channels in D–E′. Note failure of apical (luminal) localization of T1α in Dlg5^−/− cells in E′.

(F) Western blot analysis of total protein extracts from E15.5 wild-type (Ctrl) and Dlg5^−/− lungs with anti-Dlg5, anti-E-cadherin (E-cad), anti-Keratin 8 (Ker 8), anti-FoxJ1, anti-Sox2, anti-Sox9 and anti-β-actin antibodies.

(G–G′) Immunofluorescent stainings of lung sections from E15.5 wild-type (Ctrl) and Dlg5^−/− embryos with anti-desmin (green, Desm), anti-pro-SPC (red) antibodies. Western blotting on E15.5 total protein extracts with anti-Desmin and anti-β-actin antibodies is shown in the inset.

(H) qRT-PCR analysis of indicated gene expression in E13.5 wild-type (Ctrl) and Dlg5^−/− lungs. Mean values with standard deviations are in arbitrary units with the level of wild-type lung adjusted to 1. * indicates P<0.05, t-test, n=3–4 for each genotype.

(I–J′) Immunohistochemistry staining of newborn (P0) lung sections from wild-type (Ctrl) and Dlg5^−/− pups with anti-Aquaporin 5 (I–I′) (AEC1 marker at P0) and anti-proSPC (J–J′) (AEC2 marker at P0) antibodies.

(K) Quantitation of the proportion of AEC1 (Aqua5+) and AEC2 (proSPC+) cells in P0 wild-type (Ctrl) and Dlg5^−/− lungs. * indicates P<0.05. Mann-Whitney test, n=4.

(L) Western blot analysis of total protein extracts from E17.5 and P0 wild-type (Ctrl) and Dlg5^−/− lungs with anti-pro-SPC (AEC2 marker), CCSP (Clara cell marker), anti-T1α (AEC1 marker) and anti-β-actin antibodies. Note prominent increase in pro-SPC and decrease in T1α levels in Dlg5^−/− lungs.

(M) qRT-PCR analysis of indicated gene expression in P0 wild-type (Ctrl) and Dlg5^−/− lungs. ** indicates P<0.01. t-test, n=3

(N–N′) Immunofluorescent stainings of lung sections from P0 wild-type (Ctrl) and Dlg5^−/− pups with anti-CCSP (Clara cell marker) antibodies.

Bar in A represents 1mm in A–A′, 0.4 mm in B–B′, G–G′, I–J′, N–N′,100 μm in C–E′.