Fig. 5.

The interstitial axis is marked by MEOX2 and includes distal Wnt2-expressing PDGFRA-low cells. (A) scRNA-seq UMAPs of lung mesenchymal cells of the interstitial axis (cell number in parentheses), colored by developmental ages or cell types, as supported by relevant markers shown in the dot plot. Meox2 is lower in a subset of proximal interstitial cells. Distal interstitial cells form subclusters of immature (E17, E19, P7 and P13) and mature (P20 and P70) cells. (B) Left: MEOX2 is not expressed by immune cells (CD45), genetically labeled endothelial (Cdh5-CreER) or epithelial (Shh^Cre) cells. Genetic labeling is necessary to circumvent co-staining of antibodies from the same species. Right: nearest neighbor analysis of internuclear distance showing that MEOX2⁺ cells occur at a comparable distance to AT1, AT2 or other nonepithelial cells (ordinary ANOVA with Tukey test; data are mean±s.d.). See Fig. S6B for representative images. (C) MEOX2 is not expressed by PDGFRA-high alveolar myofibroblasts or PDGFRB⁺ pericytes. Higher magnification images show perinuclear distribution of PDGFRA and PDGFRB. (D) PDGFRA-low cells (1-3) have dim GFP and express MEOX2, whereas PDGFRA-high cells (4,5) have bright GFP and perinuclear PDGFRA. (E) Single-cell morphology of ten distal interstitial cells from sparse genetic labeling with 3 mg Tamoxifen 2 days before lung harvest. (F) Immunostaining of an embryonic lung shows that MEOX2⁺ distal interstitial cells are distinct from PDGFRA⁺ and PDGFRB⁺ cells surrounding the epithelium and vessels, respectively. (G) Schematic of distal interstitial cells. Created with BioRender.com. Scale bars: 10 µm. epi, epithelium; Tam, 250 µg (B) and 300 µg (C) Tamoxifen; v, vessels.