Fig 1: 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).