Tenascin-C expressing touch dome keratinocytes exhibit characteristics of all epidermal lineages

Abstract

The touch dome (TD) keratinocytes are specialized epidermal cells that intimately associate with the light touch sensing Merkel cells (MCs). The TD keratinocytes function as a niche for the MCs and can induce de novo hair follicles upon stimulation; however, how the TD keratinocytes are maintained during homeostasis remains unclear. scRNA-seq identified a specific TD keratinocyte marker, Tenascin-C (TNC). Lineage tracing of Tnc-expressing TD keratinocytes revealed that these cells maintain themselves as an autonomous epidermal compartment and give rise to MCs upon injury. Molecular characterization uncovered that, while the transcriptional and chromatin landscape of the TD keratinocytes is remarkably similar to that of the interfollicular epidermal keratinocytes, it also shares certain molecular signatures with the hair follicle keratinocytes. Our study highlights that the TD keratinocytes in the adult skin have molecular characteristics of keratinocytes of diverse epidermal lineages.

Fig. 1.. Tnc is expressed in the TD keratinocytes of adult dorsal skin.

(A) Schematic showing the Gli1^TOM+ populations that were isolated for scRNA-seq. (B) Cell transcriptomes were visualized with Uniform Manifold Approximation and Projection (UMAP) and colored according to unsupervised (first-level) clustering. (C) Expression of Krt17 and Tnc projected onto the UMAP. (D) Violin plot depicting the expression of TD signature genes in comparison to IFE basal and HF bulge cells. (E) Immunofluorescence (IF) of DAPI (blue), KRT17 (green), KRT8 (white), and in situ of Tnc RNA (red) in the TD of P52 wild-type mice. Note that Tnc RNA was detected in the TD keratinocytes, a subset of MCs and dermal cells below the TD. (F) IF of DAPI (blue), KRT5 (green), KRT8 (white), and TNC protein (red) in the TD of P52 wild-type mice. Note that TNC protein was detected only in the dermal cells below the TD. Scale bars, 50 μm for 20× and 25 μm for 40× images.

Fig. 2.. Tnc-CreER efficiently labels TD keratinocytes.

(A) Experimental design of lineage tracing in Tnc-CreER; R26^tdTomato mice. (B to D) Tnc-CreER; R26^tdTomato lineage tracing in the back skin of P60 (B), P150 (C), and P365 (D) mice, respectively. IF analysis of DAPI (blue) TOMATO (red), KRT17 (green), and KRT8 (white). Insets show a higher magnification of TOMATO expressing TD keratinocytes. (E) A scatterplot representing the average percentage ± SEM of TOMATO⁺/ KRT17⁺ cells in Tnc-CreER; R26^tdTomato back skin sections of P60, P150, and P365 mice (n = 100 TDs across three to four animals per time point). Nonparametric one-way ANOVA (Kruskal-Wallis test) was conducted. P value = 0.0619. (F) A scatterplot representing the average percentage ± SEM of EdU⁺/KRT17⁺ cells in the back skin of P21, P28, P42, P52, and P95 mice (n = 30 TDs across three to four animals per time point). One-way ANOVA (Dunnett’s multiple comparisons test) was conducted. P values = 0.0014 (P21 versus P28); 0.83 (P21 versus P42); 0.97 (P21 versus P52); 0.0031 (P21 versus P95); 0.0099 (P52 versus P95). Scale bars, 25 μm.

Fig. 3.. TNC expressing TD cells give rise to MCs upon mild injury.

(A) Whole-mount IF analysis of KRT8 (green) and Ki67 (red) in TDs of dorsal skin of unwaxed control, 24-, 48-, 72-, and 96-hour post-wax (p.w.) mice. (B) A scatterplot representing the average number of KRT8⁺ MCs in TDs of dorsal skin of unwaxed control, 24-, 48-, 72-, and 96-hours p.w. mice (n = 30 to 40 TDs across six animals per time point). One-way ANOVA (Dunnett’s multiple comparisons test) was conducted. P values = <0.0001 (unwaxed control versus 24 hours p.w.); 0.11 (unwaxed control versus 96 hours p.w.); >0.99 (24 hours p.w. versus 48 hours p.w.); 0.06 (24 hours p.w. versus 72 hours p.w.); 0.0028 (24 hours p.w. versus 96 hours p.w.). (C) IF analysis of DAPI (blue), KRT17 (green), and Ki67 (red) in the dorsal skin of unwaxed control, 24-, 48-, 72-, and 96-hours p.w. mice. (D) A scatterplot representing the average percentage ± SEM of Ki67⁺/KRT17⁺ cells in TDs of dorsal skin of unwaxed control, 24-, 48-, 72-, and 96-hours p.w. mice (n = 15 to 20 TDs across three animals per time point). One-way ANOVA (Dunnett’s multiple comparisons test) was conducted. P values = 0.085 (unwaxed control versus 24 hours p.w.); <0.0001 (unwaxed control versus 48 hours p.w.); <0.0001 (unwaxed control versus 72 hours p.w.); <0.0001 (unwaxed control versus 96 hours p.w.). (E) Experimental design of lineage tracing in Tnc-CreER; R26^tdTomato mice. The dorsal skin was waxed at P58 and collected 23 days later. (F) IF analysis of DAPI (blue), TOMATO (red), KRT17 (green), and KRT8 (white) in P80 unwaxed control and waxed skin of Tnc-CreER; R26^tdTomato mice. (G) A scatterplot representing the average percentage ± SEM of TOMATO⁺/KRT8⁺ cells in unwaxed and 23 days p.w. Tnc-CreER; R26^tdTomato mice (n = 30 TDs per group across three animals). An unpaired t test was conducted to test for significance. P value = 0.0262. Scale bars, 25 μm.

Fig. 4.. TD keratinocytes are similar to the IFE keratinocytes.

(A) Heatmap showing the correlation and clustering of the FACS-isolated IFE, TD, and HF cells from the dorsal skin of P60 Tnc-CreER; R26^tdTomato mice, based on the log-transformed read counts of all genes in RNA-seq data. RNA-seq analysis was done on three biological replicates for each group from at least two independent litters. (B) Venn diagram depicting that 175 of the 229 genes significantly up-regulated in TD when compared to IFE were also expressed more highly in HF cells. (C) Gene Ontology (GO) analysis of the 54 TD unique genes. (D) Heatmap showing the correlation and clustering of open chromatin in FACS-isolated IFE, TD, and HF cells from the dorsal skin of P60 Tnc-CreER; R26^tdTomato mice based on the log-transformed read counts at all ATAC-seq peaks. The numbers are Pearson’s correlation coefficients. ATAC-seq analysis was done on two biological replicates for each group from at least two independent litters. (E) Experimental design of long-term lineage tracing in Tnc-CreER; R26^tdTomato mice starting at P51. The dorsal skin was collected at P150 and P365. (F to H) IF analysis of DAPI (blue), TOMATO (red), KRT17 (white), and KRT5 (green) in the dorsal skin of P60 (F), P150 (G), and P365 (H) mice. White dashed lines indicate the border of KRT17⁺ TD cells. (I) A scatterplot representing the average percentage ± SEM of TOMATO⁺ TDs that have contributed to the nearby IFE (n = 60 TDs for each time point across three to four animals). One-way ANOVA (Dunnett’s multiple comparisons test) was conducted. P values = 0.65 (P60 versus P150); <0.0001 (P60 versus P365); <0.0001 (P150 versus P365). Scale bars, 25 μm.

Fig. 5.. TD keratinocytes share molecular characteristics with the HF.

(A) Volcano plot depicting the differentially expressed genes in FACS-purified TD versus IFE cells from the dorsal skin of P60 Tnc-CreER; R26^tdTomato mice. Genes with absolute fold change > 1.5 and adjusted P value < 0.05 were considered significantly up-regulated or down-regulated in TD cells. (B) RT-qPCR analysis of HF genes that were identified to be expressed higher in TD cells than in IFE via RNA-seq and which have greater chromatin accessibility in HF and TD than in the IFE. (C) IF analysis of DAPI (blue) and HFSC marker LHX2 (red) in TD which was identified by the presence of KRT8 (green) expressing MCs. LHX2 single channel is shown in gray. Scale bar, 10 μm. (D) Scatterplot comparing the degrees of chromatin accessibility changes in TD and HF versus IFE, revealing that of 1021 ATAC-seq peaks that are significantly more accessible [false discovery rate (FDR) < 0.05 and fold change (FC) >1.5] in the TD than in the IFE, 881 of them are also significantly greater in HF, including peaks at some key HF genes (green dots). (E) IGV tracks of ATAC-seq profiles at selected HF signature genes in FACS-isolated IFE, HF, and TD keratinocytes.