Figure 4.

Embigin deficiency delays embryonic lung development

(A) Representative images of hematoxylin-eosin-stained lung sections from two WT and two Emb^−/− E17.5 littermates are presented. Scale bars: 50 μm. The relative area of small airways and distal airspaces in the E17.5 lung sections was analyzed by ImageJ/Fiji software (n = 11 for WT and n = 14 for Emb^−/−). Statistical significance (p = 0.00002) was determined by Mann-Whitney U-test. Data are represented as a Spear style boxplot. A square shows the mean value.

(B) The correlation between the body weight of the embryo and the relative area of small airways and distal airspaces in the lung sections was studied by calculating Spearman’s rank correlation coefficient (r_s) and its statistical significance.

(C) Alkaline phosphatase activity was determined from the amniotic fluids of WT, Emb^+/−, and Emb^−/− embryos at E17.5 with VetScan Chemistry Analyzer (n = 6 for WT, and n = 5 for Emb^+/− and Emb^−/−). Statistical significance (p = 0.024) was determined by ANOVA followed by Dunnett’s t-test. Data are represented as a Spear style boxplot. A square shows the mean value.

(D) Lung explants from E12.5 WT and Emb^−/− embryos were cultured for 2 days and imaged on days 0, 1, and 2 (D0 – D2, n = 14 for WT and n = 10 for Emb^−/−). The normalized count of peripheral buds in lung explants of WT and Emb^−/− at E12.5 was compared at D0, D1, and D2 (normalization: value/biggest value of the litter at D0). Statistical significances (D0: p = 0.00002, D1: p = 0.002, and D2: p = 0.034) were determined by Student’s T-test for independent samples. Data are represented as a Spear style boxplot. No statistical difference between WT and Emb^−/− was observed from D0 to D2 in the progression of branching morphogenesis. Scale bars: 1000 μm.

(E) WT and Emb^−/− lung paraffin sections were immunostained with SPC and HOP antibodies at E17.5 and nuclei were labeled with Hoechst 33342 Fluorescent Stain. The number of SPC (ATII) and HOP (ATI) -positive cells in WT (n = 6) and Emb^−/− (n = 7) embryonic lungs at E17.5 were counted from five different areas of each lung. The amounts of SPC-positive cells in each sample were statistically compared with Mann-Whitney U-test (p = 0.022) and HOP-positive cells were analyzed by Student’s T-test for independent samples (p = 0.005). Data are represented as a Spear style boxplot. Scale bars: 100 μm.

(F) A representative transmission electron micrograph of an alveolar ATII cell from Emb^−/− lungs at E18.5 is presented. Black arrows point to mature lamellar bodies and arrowheads to multilamellar bodies, N: nucleus; M: mitochondria, MVB: multivesicular body. Scale bar: 2000 μm. The amounts and the sizes of lamellar bodies of ten ATII cells in each sample were analyzed (n = 3 for WT and Emb^−/−), and medians (n = 71–96/sample) were statistically analyzed by Student’s T-test for independent samples. The medians of each sample and mean ± SD are shown.

See also Figures S3 and S5.