Figure 2. A first wave generates both epithelial compartments.

(a) Optical projection tomography (OPT) images of whole mount immunostained lungs. All whole embryonic lungs and the P18 left lobe are shown to the same scale (Scale: 500 um) with the exception of the E10 foregut and E11 lung, which are shown at a higher magnification (Scale: 100 um). In a side view of the E10 foregut, the nascent left lung bud (left) is outlined with a dashed line. The L.L2 branch lineages ¹⁴ are traced with dashed lines, and shown as inset for the P18 lung and separately for the embryonic lungs (right panels, Scale: 500 um). Double-sided arrows indicate that the gap between the distal boundaries of the SOX2 and SOX9 expression domains widens after E17. Due to variation in the branch lineage ¹⁴, BADJ formation (closed arrowheads) are inferred by counting branches (b) and from lineage tracing experiments. Asterisks indicate background staining from blood vessels only present in older postnatal lungs. Lower left panels (Scale: 100 um): projection of confocal images of immunostained E17 and E19 lung lobe edges for a high resolution view of SOX9 positive branch tips (dashed line). (b) Quantification of the number of SOX9 positive branch tips (green circle) and SOX2 positive branch termini (red cross) of the planar bifurcation of L.L1 through L.L6 branch lineages ¹⁴. The number of SOX2 positive termini plateaus at E17 (closed arrowhead). The number of SOX9 positive tips reaches the maximal number of SOX2 positive termini at E15 (open arrowhead). (c, e) Whole mount in situ hybridization of lungs at E14 (early branches) and E17 (late branches) for markers of simple epithelium (Krt8, Krt18, (c)) and branching related genes identified in an in silico screen of Eurexpress ⁶⁹ (e). Branch tips are outlined with dashed lines. Scale: 20 um. (d) Confocal images of whole mount immunostained lungs at E14 (early branches) and E17 (late branches). pERK, phosphorylated Extracellular signal-Related Kinase. pERK-positive branch tips are outlined with dashed lines. Cells in the vasculature are also pERK positive (arrowheads). Scale: 20 um.