Extended Data Fig. 2: In situ RNA hybridization with amplification confirms the postnatal disruption of spatial organization between different cell types in NDUFS2 cKO lungs.

a–b, Lung sections from 21-day-old NDUFS2 control and NDUFS2 cKO mice (n = 3 each genotype, both male and female) were hybridized with the indicated target probes (RNAscope®). Magnified images (boxed region) are shown on the right. Representative lung sections (a) showing alveolar type 2 (AT2) cells by Sftpc (gray) have 1:1 direct contact with Pdgfra⁺ fibroblasts (magenta) in NDUFS2 control lungs, whereas 1:1 relationship between Sftpc⁺ cells and Pdgfra⁺ fibroblasts are lost in NDUFS2 cKO lungs. Hypertrophic Sftpc⁺ cells in NDUFS2 cKO lungs cluster next to each other along the alveolar walls while Sftpc⁺ AT2 cells in NDUFS2 control lungs locate individually at the corner of alveolar sacs. Representative lung sections (b) showing Car4⁺ endothelial cells (cyan, arrows) locate next to linear thin Sftpc- AT1 cells in NDUFS2 control mice. However, in NDUFS2 cKO lungs, Car4⁺ endothelial cells (cyan) are located next to linear thin Sftpc⁺ cells (gray, asterisk). Please note that Sftpc is an AT2 marker (cuboidal) that does not normally express in linear thin AT1 cells. Scale bars, 50 μm and 20 μm (magnified inset). c–g, Mitochondrial complex I in lung epithelial cells is dispensable for antenatal lung development. Representative images of littermates’ lung histology (hematoxylin-eosin stain) at different time points (E, embryonic day; P, postnatal day). Branching morphogenesis during antenatal development is not grossly disrupted in NDUFS2 cKO mice compared to NDUFS2 control mice (c,d). The subtle differences in alveolar structure between NDUFS2 cKO and NDUFS2 control mice at P11 become apparent by P21 (e–g). Scale bars, 200 μm (c,d), 50 μm (e–g).