Platelets are required for lung development. (A) Clec-2 expression on platelets in E17.5 Clec1b^fl/fl (wild-type), Clec1b^ΔPLT and Clec1b^−/− fetuses. Gray-filled and open histograms indicate isotype control and Clec-2, respectively. (B) Platelet counts at E17.5 in Clec1b^fl/fl and Clec1b^ΔPLT fetal blood treated with rat IgG (control), anti-Clec-2 antibody, or anti-GPIb antibody. Mean ± SD, n = 3, 4, 4, 3, 3, and 4, respectively, from the left of the histogram. N.S., not significant, Tukey’s test. (C) Neonatal lethality in Clec1b^fl/fl and Clec1b^ΔPLT neonates treated with rat IgG (control), anti-Clec-2, or anti-GPIb. *P = .0021, **P = .0237, Fisher’s exact test. (D) Immunostaining of α-SMA (green) and DAPI (blue) in E17.5 distal lung. AL, alveolar sac; M, lung mesothelium. (E) Quantification of α-SMA expression in E17.5 distal lung. Asterisks indicate the 2 groups in which α-SMA expression was significantly lower than that in the other 4 groups; mean ± SD, n = 3 each; *P < .005, Holm-Sidak test. (F) Whole-mount immunohistochemistry of Wt1 (magenta) and Pdpn (green) in E17.5 luMCs. Arrowheads: Wt1-negative luMCs. (G) Quantification of Wt1-negative rate in luMCs; mean ± SD, n = 3 each. *P = .0019; **P = .0022, Tukey’s test. (H) Quantification of luMC density; mean ± SD, n = 3 each. *P = .0034; **P < .0001, Tukey’s test. (I) Correlation between Clec-2 expression in E17.5 fetal platelets (supplemental Figure 3) and neonatal lethality in the listed groups (Figures 1A,4C, and5C and Clec1b^ΔPLT/−); mean ± SD of Clec-2 expression level is shown. Numbers of individuals in all groups are shown in supplemental Figure 3. A very strong correlation (R² = 0.8783) was observed in the 6 experimental sets of mice exhibiting more than 10% neonatal lethality (right side of the dashed line). Neonatal lethality of Clec1b^ΔPLT/− was 50.0% (5/10). Scale bars: 25 μm (D,F).