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. 2022 Nov 10;24(12):1692–1700. doi: 10.1038/s41556-022-01021-8

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© The Author(s) 2022

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. 5 — (A) Classification of cells from all datasets as Krt10-positive or Krt10-negative (cutoff: 1.84 log-normalized and downsampled counts, see Methods). Cells are colored according to their Krt10 expression levels. (B) Quantification of Krt10-positive and negative cells within the basal and suprabasal compartments (as defined by the delamination point). (C) Mapping of (Aragona et al., 2020) dorsal IFE dataset (colored) onto our combined UMAP (in grey). Colors indicate cluster annotations from Aragona dataset. (D) UMAP showing the grouping of cells into bins according to their location along the differentiation pseudotime. The basal-suprabasal border was set between bins 6 and 7 (Methods). (E) Expression patterns of genes shown in Fig. 2F, overlaid on the combined UMAP. (F) Violinplots grouped according to pseudotime bins (left panel) and combined UMAPs (right panel) showing characteristic gene expression changes during the differentiation process. Expression levels are shown as log-normalized expression. (G) smRNA-FISH validation of differentiation-associated gene expression in basal dorsal IFE cells. Arrowheads indicate basal cells with Krt10⁺ /Krtdap⁺ (red) or Krt10⁺ /Krtdap⁻ (white) expression. (H-I) smRNA-FISH and antibody-based stainings showing basal Ivl-traced cells (Tom⁺ stained via RFP) with Krt5 and Mt4 co-expression (arrowheads) (H), and with Krt14 and Krt10 co-expression (arrowheads) (I). (G-I) Cell membranes are stained with WGA (wheat germ agglutinin). Dashed lines indicate the basement membrane. Scale bars = 25 μm. (A, C, D, E, F) Dashed lines indicate the assigned basal-suprabasal border. (J) Representative whole mount staining showing K10 (red) and K1 (green) protein overlap in the basal layer of dorsal epidermis. Cell boundaries are visualized with phalloidin (white). Insets highlight an example of K10 positive, K1 low cells (arrowhead). Scale bar = 25 µm (large field of view) or 10 µm (inset). Images in (G-J) are representative of staining from n = 3 mice. (A-F) Plots show integrated results of all biological replicates from all datasets combined (Joost 2016 n = 19, ITGA6-sorted n = 2, Joost 2020 n = 5 mice) with exception of (C) which includes additional integration of Aragona dataset.