Fig 2: Defining distinct epithelial subclusters in PN14 EPCs(A) tSNE representation of reclustered EPCs data based on Sfptc expression, showing eight distinct subclusters, C4 to C11, in both control and mutant lungs.(B) Alterations in cell number in each of the eight subclusters as a fraction of total lung cells, or total epithelial cells between control and mutant lungs. PS, population shift.(C and D) Heatmaps of enriched transcripts in each of the control and mutant subclusters and their overlap with alveolar epithelial cell lineage signature genes derived from LungGens Consortium.(E) tSNE representation of select enriched transcripts for C4 (Mki67, Top2a, Cdk1, and Spc24) and their violin plot abundance and differential expression in C4 to C11.

Fig 3: Representative images showing many higher numbers of proliferating and KRT14+ urothelial cells in Vehicle 1 (Veh1)-treated versus FGF7p-treate mice 28 days after cyclophosphamide. (a–f) Triple label IF for Ki67 (red), KRT14 (white), and KRT5 (green) in Vehicle 1-treated (a–c) and FGF7p-treated (d–f) mice. (a, d) IF for Ki67 with DAPI (blue) shows that the Vehicle 1-treated mouse (a) has many proliferating urothelial cells, with the majority found in basal layers (arrow) while the FGF7p-treated mouse (d) has significantly fewer proliferating urothelial cells that are also primarily in basal layers (arrow). (b, e) Merged Ki67 and KRT14 staining shows that both the vehicle 1-treated mouse (b) and the FGF7p-treated mouse (e) have many KRT14+ basal cells that are proliferating (arrows) and other KRT14+ cells that are not proliferating (arrowheads), although the Vehicle 1-treated has many more of each than the FGF7p-treated mouse. (c, f) Merged Ki67 and KRT5 staining shows that in the both the Vehicle 1-treated mouse (c) and the FGF7p-treated mouse, all of the KRT14+ cells that are proliferating (arrows) or not proliferating (arrowheads) are KRT5+, although some of the proliferating KRT5+ cells are KRT14- (concave arrowheads). L (a–f) = lumen. Dashed line (a, d) = urothelial-stromal boundary. Scale bars (a–f) = 50 µm. (g) Graph showing that the mean percentage of total Ki67+ proliferating urothelial cells and mean percentage of KRT14+ proliferating urothelial cells are higher in the Vehicle 1-treated mice than FGF7p-treated mice 28 days after injury; there are no differences in percentages of KRT14- proliferating urothelial cells between the two groups. N = 6 (3 bladders with two planes assessed per mouse). **p < 0.01. (h) Graph showing that the mean percentage of KRT14+ cells is higher in the Vehicle 1-treated mice than the FGF7p-treated mice 28 days after injury. N = 6 (three bladders with two planes assessed per mouse). **p < 0.01.

Fig 4: Functional examination of candidate ligands in 3D organoid cultures and localization of Wnt2-expressing cells in PN14 lungs(A) GFP(+) pAT2s were isolated from Sftpc-Gfp PN14 lungs and cultured with PN14 mesenchymal cells as described in STAR Methods. 3D cultures were seeded with 1,250 GFP(+) pAT2 cells and 50K mesenchymal cells and were examined on day 28 for direct GFP(+) cells. Ligand-receptor interactions between pAT2 and mesenchymal cells identified in Figure 5 were functionally examined by inhibiting the indicated pathways as shown. Inhibitors were used to block HGF (capmatinib, 1.0 µM), BMP (recombinant GREM2, 100 ng/mL), NOTCH (DAPT, 25 µM), FGF (BDJ398, 1 µM), WNT (XAV939, 1 µM) and IGF1 (PPP, 4 µM). (a1-a7) Photomicrographs of representative organoids in each culture. (a8) quantification of the number of clones (clonogenicity). (a9) Quantification of organoid size. Scale bar, 1mm (a1-a7).(B). (b1 and b2) tSNE representation of Wnt2 and Fgfr2 transcript distribution in control lung. (b3–b8) RNAscope analysis showing DAPI alone (b3), Mki67 (b4), Fgfr2 (b5), Wnt2 (b6), Fgfr2/Mki67 merge (b7), and Wnt2/Mki67 merge (b8). Red arrows show Wnt2(+) mesenchymal cells juxtaposed to Mki67(+) pAT2 cells, shown by green arrows. Scale bar, 10 µm (b3–b8).(C) Modeling impact of cell population changes on neonatal pAT2 self-renewal. Different ratios of GFP(+) pAT2 cells and mesenchymal cells from PN14 control lungs were cultured as described in STAR Methods. Cell ratios are shown above each panel. 3D cultures were examined on day 21 for direct GFP(+) cells. (c1–c6) Increasing number of GFP(+) pAT2 cells cultured with 50K mesenchymal cells. (c7–c12) 5K GFP(+) pAT2 cells cultured with increasing number of mesenchymal cells as shown. (c13 and c15) Quantification of the number and size of the colonies respectively formed in c1–c6. (c14 and c16) Quantification of the number and size of the colonies, respectively formed in c7–c12. For each culture, n = 3. Scale bar, 1 mm (c1–c12). Data are presented as boxplots with n=4 (a8) and n=6 (c13,c14).

Fig 5: Dividing (KI67+) microglia had less complex, activated morphologies compared to microglia that were not dividing (KI67–). (A) Z-stacked photomicrographs of Iba1 (green) stained microglia and KI67 (red) stained dividing cells in the ipsilateral perirhinal cortex, CA1, and cortex/injection site in WT and TNF-a KO mice. (B) Total number of branches per microglia. (C) Total branch length per microglia. (D) Total number of endpoints per microglia. (E) Density of pixels per microglia. (F) Fractal dimension (Db) of microglia. (G) Circularity of microglia. (H) Lacunarity of microglia. (I) Span ratio of microglia. Individual data points are presented as dots and were analyzed using linear mixed models (see Tables 4, 5). Results are presented as point estimates with 95% confidence intervals. Statistically significant post hoc comparisons are indicated by bars with corresponding p-values. Iba1+/KI67+ = colocalization of Iba1 and KI67. TNF-a KO KI67– n = 181 microglia; WT KI67– n = 139 microglia; TNF-a KO KI67+ n = 87 microglia; WT KI67+ n = 71 microglia. Scale bar = 50 µm.