Fig. 1: Emergence and patterning of nascent AT2 cells.

a scRNAseq UMAP generated from a published timecourse study (GSE119228)³⁵. b Leiden clustering identifies mature AT2s (mAT2), nascent AT2s (nAT2), distal progenitors (DP), and AT1s. c Cluster proportion of AT2 lineage across development shows their separation over time. d Venn diagram indicates the number of genes shared or restrictively expressed. e Dot plot of stage-restricted marker gene Retnla (nAT2) and Cd74 (mAT2). f E17.5 immunostain of RELMα (green), CD74 (red), and DP/AT1-restrictive luminal marker PDPN (white). Bar, 25 µm. g E15.5 (left) and E16.5 (right) immunostain for smooth muscle actin (SMA, red), RELMα (green), E-cadherin (ECAD, blue), and PDPN (white). Close-up images for E15.5 lungs of rare single nAT2s (asterisk) emerging at intermediate regions, not terminal end buds (arrowheads). Bars, 100 µm (left and right panels), 20 µm (close-ups). Mesothelium is marked with white dash. h Quantification of RELMα⁺ nAT2 immunostains from E15.5 to E17.5 (n represents branches scored per timepoint; data are represented as median with whiskers extending to the upper and lower quartiles. p values determined using Kruskal–Wallis test). i Quantification of RELMα⁺ nAT2 morphology (columnar versus basally extruded). Extruded values presented as mean ± SD (n represents cells scored at each timepoint). j Schematic of the timing and sequence of nAT2 emergence. At E15.5 the first nAT2 emergent zone (bright green) occurs between the terminal end bud and stalks of the undifferentiated distal epithelium (blue), which ~E16.5 expands during branching morphogenesis. Around E17.5, nAT2s next emerge more proximally (dark green) as airway smooth muscle remodels into myofibroblasts—leaving the final emergence of nAT2s to occur at the distal tips after birth. Source data are provided as a Source Data file.