Fig 2: Smo mediates HH-signaling in tracheal epithelial cells and controls their differentiation and proliferation. (A) Immunostaining for FOXJ1 (red), NKX2.1 (green) and DAPI staining (blue) of longitudinal sections of E18.5 control (n=6) and SmoCKO (n=6) tracheae. (B) Quantification of the relative number of FOXJ1+ cells in the tracheal epithelium in E18.5 control (n=6) and SmoCKO (n=6) mice. (C) RT-qPCR analysis of Foxj1, Tubb4b and Scgb1a1 mRNA levels in E18.5 control (n=5) and SmoCKO (n=5) tracheae. (D) Immunostaining for acetylated a-tubulin (red), NKX2.1 (green) and DAPI staining (blue) of longitudinal sections of E18.5 control (n=6) and SmoCKO (n=6) tracheae. (E) Quantification of the relative number of acetylated a-tubulin+ cells in the tracheal epithelium in E18.5 control (n=6) and SmoCKO (n=6) mice. (F) Immunostaining for SCGB1A1 (red), NKX2.1 (green) and DAPI staining (blue) of longitudinal sections of E18.5 control (n=6) and SmoCKO (n=6) tracheae. (G) Quantification of the relative number of SCGB1A1+ cells in the tracheal epithelium in E18.5 control (n=6) and SmoCKO (n=6) mice. (H) EdU fluorescence (red), immunostaining for CDH1 (green) and DAPI staining (blue) of longitudinal sections of E18.5 control (n=6) and SmoCKO (n=6) tracheae after a 21 h 10 µM EdU treatment. (I) Ratio of CDH1+ cells that are EdU+. (J) Immunostaining for acetylated a-tubulin (red) and DAPI staining (blue) of longitudinal sections of P9 control (n=4) and Nkx2.1Cre;R26SmoM2 (n=4) tracheae. (K) Quantification of the relative number of acetylated a-tubulin+ cells in the tracheal epithelium in P9 control (n=4) and Nkx2.1Cre;R26SmoM2 (n=4) mice. (L) Immunostaining for SCGB1A1 (red) and DAPI staining (blue) of longitudinal sections of P9 control (n=4) and Nkx2.1Cre;R26SmoM2 (n=4) tracheae. (M) Quantification of the relative number of SCGB1A1+ cells in the tracheal epithelium in P9 control (n=4) and Nkx2.1Cre;R26SmoM2 (n=4) mice. Scale bars: 20 µm. Unpaired Student's t-test. Data are mean±s.d.

Fig 3: Nkx2.1Cre;Shhflox/flox mice exhibit fracture of the tracheal cartilage, pulmonary hemorrhage and cystic lungs. (A,B) mRNA in situ detection of Cdh1 (dark green), Trp63 (pink), Shh (magenta), Smo (yellow), Ptch1 (cream), Ptch2 (blue), Hhip (pale green), Gli1 (orange), Gli2 (cyan) and Gli3 (khaki) expression and DAPI staining (gray) of longitudinal sections of wild-type tracheae at different developmental stages. The dashed square in the left-hand images indicates the area enlarged and rotated in the right-hand images. Dashed lines in the right-hand images indicate the border between the tracheal epithelium and mesenchyme. (C) Quantification of the percentage of positive cells within each cell state that express HH signaling components. (D) Quantification of the percentage of positive cells that express HH signaling components. Number of cells at E12.5=239, E13.5=455, E14.5=1232, E15.5=931, E16.5=365 and E18.5=286. (E) Representative gross morphology of P0 control (n=6) and ShhCKO mutants (n=6). (F) Quantification of P0 control (n=6) and ShhCKO mutant (n=6) respiratory rates. (G) Representative images of transverse sections of tracheae stained with Alcian Blue and nuclear Fast Red from P0 control (n=5) and ShhCKO mutants (n=5). (H) Quantification of P0 control (n=5) and ShhCKO mutant (n=5) tracheal lumen areas. (I) Representative images of ventral views of whole-mount tracheae stained with Alcian Blue from P0 control (n=6) and ShhCKO mutants (n=6). Arrows indicate tracheal cartilage rings. (J) Quantification of the number of intact tracheal cartilage rings from P0 control (n=6) and ShhCKO mutants (n=6). (K) Representative images of ventral views of the trachea and lungs from P0 control (n=6) and ShhCKO mutants (n=6). White arrow indicates pulmonary cysts. Blue arrow indicates blood spots. (L) Percentage of lungs with hemorrhage. (M) Percentage of lungs with cysts. Scale bars: 2 mm in K; 1 mm in E,I; 400 µm in G; 100 µm in low-magnification tracheal large images in A,B; 10 µm in high-magnification tracheal images in A,B. Unpaired Student's t-test. Data are mean±s.d. (F,H,J,L,M). E, epithelium; M, mesenchyme; Shh, sonic hedgehog; Smo, smoothened; ShhCKO, sonic hedgehog conditional knockout.

Fig 4: Nkx2.1Cre;Shhflox/flox mice exhibit defects in tracheal epithelial cell proliferation and differentiation. (A) Immunostaining for FOXJ1 (red), NKX2.1 (green) and DAPI staining (blue) of longitudinal sections of E18.5 control (n=6) and ShhCKO (n=6) tracheae. (B) Quantification of the relative number of FOXJ1+ cells in the tracheal epithelium in E18.5 control (n=6) and ShhCKO (n=6) mice. (C) RT-qPCR analysis of Foxj1, Tubb4b, Scgb1a1 and Foxp1 mRNA levels in E18.5 control (n=5) and ShhCKO (n=5) tracheae. (D) Immunostaining for acetylated a-tubulin (red) and DAPI staining (blue) of longitudinal sections of E18.5 control (n=6) and ShhCKO (n=6) tracheae. (E) Quantification of the relative number of acetylated a-tubulin+ cells in the tracheal epithelium in E18.5 control (n=6) and ShhCKO (n=6) mice. (F) Immunostaining for SCGB1A1 (red), NKX2.1 (green) and DAPI staining (blue) of longitudinal sections of E18.5 control (n=6) and ShhCKO (n=6) tracheae. (G) Quantification of the relative number of SCGB1A1+ cells in the tracheal epithelium in E18.5 control (n=6) and ShhCKO (n=6) mice. (H) Immunostaining for CDH1 (red), Ki67 (green) and DAPI staining (blue) of longitudinal sections of E18.5 control (n=6) and ShhCKO (n=6) tracheae. (I) Ratio of CDH1+ cells that are Ki67+. (J) EdU fluorescence (red), immunostaining for CDH1 (green) and DAPI staining (blue) of longitudinal sections of E18.5 control (n=6) and ShhCKO (n=6) tracheae. (K) Ratio of CDH1+ cells that are EdU+. (L) Immunostaining for SCGB1A1 (red), Ki67 (green) and DAPI staining (blue) of longitudinal sections of E18.5 control (n=6) and ShhCKO (n=6) tracheae. (M) Ratio of SCGB1A1+ cells that are Ki67+. Scale bars: 20 µm. Unpaired Student's t-test. Data are mean±s.d.

Fig 5: TypeTracker method overview and primary data.(A) Method workflow. Step 1: Image several organoids in 3D for 60 hours. Step 2: Track all cell trees using convolutional neural network. Each cell is uniquely identified from frame to frame, yielding a lineage (black line). Division events give a fork-like structure. Step 3: Identify cell type at movie end point using multiple rounds of antibody staining. Step 4: Map cell types onto lineage tree and infer historical cell types including type transitions (triangles). (B) Reconstructed cell positions at the start of the lineage tree. (C) Example lineage trees and their mapped end point cell types. Numbers, lineages of three cells seen in (B); stars, end point sister pairs of same type but of different type than cousins, suggesting type transition in mother cell. (D) Nuclear marker (magenta) and antibody or dye staining (green). Olfm4, olfactomedin 4; WGA, wheat germ agglutinin; Cga, chromogranin A; Lyz, lysozyme; Aldoß, aldolase ß; KRT20, cytokeratin 20; Ecad, E-cadherin. Scale bar, 10 µm, except for the Ecad panel (20 µm). (E) Cell type identification table. (F) Reconstructed cell positions and type at end of the lineage tree (C) and 66 hours after the start (B).