Clec1b deficiency causes lung mesothelial cell hyperproliferation and abnormal differentiation. (A) Lineage tracing of adMYFs in E17.5 Wt1-EGFPCre, CAG-CAT-EGFP lung sections by α-SMA (magenta) and EGFP (green) staining and DAPI (blue) counterstaining; magnified views of framed areas in images are shown on the right. Arrowheads indicate α-SMA⁺EGFP⁺ cells (yellow), α-SMA⁺EGFP⁻ cells (white). (B) Immunostaining of BrdU (red) and Pdpn (green) and DAPI staining (blue) of E17.5 distal lung sections. Pdpn immunostaining distinguishes the mesothelium, epithelium, and mesenchyme, as Pdpn is expressed in luMCs, AECs, and LECs in the distal lung. Arrowheads: BrdU-positive mesothelial cells (yellow), epithelial cells (black), and mesenchymal cells (white). (C) Histogram showing BrdU-positive rates in mesothelial, epithelial, and mesenchymal cells in E17.5 distal lung; mean ± SD, n = 3 each; *P = .0014, Holm-Sidak test. (D) TUNEL (red) and DAPI (blue) staining of E17.5 distal lung sections. TUNEL-positive apoptotic cells were very rare in both Clec1b^+/+ and Clec1b^−/−. (E) Whole-mount immunohistochemistry of Wt1 (magenta) and Pdpn (green) in E17.5 luMCs. Arrowheads: Wt1-negative luMCs. (F) Quantification of Wt1-negative rate in luMCs at E15.5, E16.5, and E17.5; mean ± SD, n = 3 each. *P < .0001; **P = .0074, Tukey’s test. (G) Quantification of luMC density at E15.5, E16.5, and E17.5; mean ± SD, n = 3 each. *P = .0002; **P < .0001; ***P = .0010, Tukey’s test. AL, alveolar sac; M, lung mesothelium. Scale bars: 25 μm (A-B,D,E).