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. 2023 Jan 16;25(2):351–365. doi: 10.1038/s41556-022-01064-x

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 8 — (a) Balloon-plot of known epithelial markers in the clusters of Fig. 4a, using data from all analyzed donors. General: EPCAM, CDH1, Proximal: SOX2⁶, Ciliated: FOXJ1¹⁰⁷, Neuroendocrine: CHGA, ASCL1¹⁰⁶, Basal: TP63, KRT5¹³⁷, Club cells: SCGB1A1, SCGB3A2¹³⁸, Distal: SOX9⁶, FGF20, Alveolar Type 1 (AT1): HOPX, PDPN, AQP5⁶, AT2: SFPTC, ETV5¹³⁹ and Proliferating: MKI67¹¹⁴, PCNA¹²⁰ together with the top-5 identified selective markers (adjusted p-value <0.001, MAST, Bonferroni corrected). The top-10 (log2 fold-change) were selected according to the percentage of positive cells in the cluster. The top-5 were plotted. Gene order follows the cluster order. (b) Annotation of segmented airway areas with PciSeq, using HybISS data in 5.5 pcw (left) and 13 pcw (right) airways. Distal clusters: cross, proliferating: inverted triangle and proximal: circle. Gray arrows: prox. progenitor2 (cl-4), magenta arrowheads: CTGF^high distal (cl-3). ‘prox.’: proximal, ‘arw’: airway. (c) PAGA-plot of the analyzed epithelial cells, superimposed on the Fig. 4a UMAP-plot. Line thickness: cluster-connection probability. (d) Epithelial-cell scVelo-analysis. Arrow direction: future cell-state, arrow size: transition possibility. (e) Balloon-plot of known embryonic basal-cell markers⁴⁷. (f) Balloon-plot of the top-20 adult basal-cell markers², together with TP63 expression in our dataset (blue) shows minimal expression of typical adult basal-cell markers in epithelial cells. (g) Single-plane confocal-microscopy immunofluorescence images for TP63 (magenta), KRT5 (cyan) and E-cadherin (yellow) on 8.5 (top) and 14 (bottom) pcw lung sections. TP63^pos cells were mainly localized in proximal airways, with a very small portion being KRT5^pos. Nuclear DAPI: gray. Scale-bar: 10 µm. In Balloon-plots, balloon size: percent of positive cells. Color intensity: scaled expression. Blue: high, Gray: low.