Volume 25, Issue 3, Pages 585-597.e7 (October 2018) Single-Cell Transcriptomics of Traced Epidermal and Hair Follicle Stem Cells Reveals Rapid Adaptations during Wound Healing  Simon Joost, Tina Jacob, Xiaoyan Sun, Karl Annusver, Gioele La Manno, Inderpreet Sur, Maria Kasper  Cell Reports  Volume 25, Issue 3, Pages 585-597.e7 (October 2018) DOI: 10.1016/j.celrep.2018.09.059 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2018 25, 585-597.e7DOI: (10.1016/j.celrep.2018.09.059) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 Single-Cell RNA-Seq of Traced Lgr5 and Lgr6 Cells in Wound Healing (A) Summary of the experimental workflow, highlighting the contribution and analysis of Lgr5-traced cells during the wounding process (Lgr6-traced cells were processed in the same way). In brief, tracing was started 1 week prior to wounding of dorsal epidermis (2nd telogen). Epidermal cells from the wound area and an unwounded control area of Lgr5-EGFP-Ires-CreERT2/R26-tdTomato (Lgr5TOM) and Lgr6-EGFP-Ires-CreERT2/R26-tdTomato (Lgr6TOM) mice were isolated at the given time points after wounding for sequencing and imaging, respectively. Fluorescence-activated cell sorting (FACS)-sorted Tomato+ cells were sampled and sequenced at random, and wound cells were identified computationally (STAR Methods). (B) Representative cross-sections of wounds taken at the given time points after wounding showing the temporospatial distribution of Lgr5TOM and Lgr6TOM cells. Dotted lines: approximate location of the basement membrane (thin) and wound front (thick). Filled lines: approximate location of the skin surface. Ep, epidermis; LE, leading edge; WEp, wound epidermis; WF, wound front. Scale bars: 100 μm (panoramas); 50 μm (zoom-ins). (C) Projection of Lgr5TOM and Lgr6TOM control cells onto t-SNE space defined by epidermal basal cells in telogen (Joost et al., 2016). (Left) t-SNE plot taken from Joost et al. (2016) showing the heterogeneity of epidermal basal cells. (Right) Projection of traced Lgr5TOM and Lgr6TOM (0-day control cells) onto the t-SNE map confirmed accurate cell identities of sampled cells. Immunofluorescent images show the location of Lgr5TOM and Lgr6TOM cells in the tissue. Dotted lines: approximate location of the basement membrane. Filled lines: approximate location of the skin surface. HF, hair follicle; IFE, interfollicular epidermis. Scale bars: 25 μm. (D) Histograms with transcriptome diversity score D (defined as the sum of negative logarithmic expression probabilities in reference to homeostatic bulge cells) for Lgr5TOM cells from 0-day control, unwounded control area, wound area, and inferred wound cells. Red lines demarcate the selection threshold. LOOCV, leave-one-out-cross-validation. (E) Histograms with transcriptome diversity score D (defined as the sum of negative logarithmic expression probabilities) for Lgr6TOM cells from 0-day control, unwounded control area, wound area, and inferred wound cells. Red lines demarcate the selection threshold. Cell selection was performed both in reference to homeostatic IFE cells (left panel) and homeostatic isthmus cells (right panel). Asterisk: 116 Lgr6TOM cells qualified as wound cells when compared to IFE, 184 Lgr6TOM cells when compared to IST, and 106 Lgr6TOM cells when compared to both (only those were considered for further analysis). See also Figure S1 and Table S1. Cell Reports 2018 25, 585-597.e7DOI: (10.1016/j.celrep.2018.09.059) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 Transcriptional Heterogeneity of Lgr5 and Lgr6 SC Progeny in Wound Healing (A) Wound cells group into discrete clusters. Shown are Lgr5TOM (left) and Lgr6TOM (right) cells projected onto a t-SNE using 1,000 variable genes each. Cells were colored according to unsupervised clustering, with similar clusters being grouped into more general wound cell states. Colors and labels highlight similar states present in both Lgr5TOM and Lgr6TOM wound cells. Note that naming (A/B) within cell state 1 does not necessarily imply a temporal order but is instead based on the expression of the early wound signature (state 1A expresses signature at intermediate level and state 1B at peak level). (B) Contribution of sampling time points to Lgr5TOM (left) and Lgr6TOM (right) wound cell states, with crosses denoting the median per wound state. (C) Barplots showing the (size-factor normalized) expression of selected marker genes in Lgr5TOM (left) and Lgr6TOM (right) wound and control cells, ordered and colored according to wound states defined in (A). Lines depict mean gene expression for each state. See also Figures S2 and S3 and Table S2. Cell Reports 2018 25, 585-597.e7DOI: (10.1016/j.celrep.2018.09.059) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 Rapid Activation and Niche-Signature Alteration in Lgr5TOM Cells upon Wounding (A) Projection of Lgr5TOM (control cells and states 0, 1, and 2 cells; in blue) and Lgr6TOM (state 1 cells; in orange) cells onto a t-SNE map using bulge and IFE basal marker genes. The green and blue shadings mark the distribution of telogen IFE basal cells and outer bulge cells in non-wounding conditions (data from Joost et al., 2016). (Lower panel) Cells were grouped into bins based on their position along the arrow (representing a pseudotime), reflecting their degree of bulge or IFE niche signature gene expression. (B) Expression of outer bulge marker genes (OB > IFE in telogen dataset with 99.9% posterior probability [PP]) in Lgr5TOM cells, ordered by pseudotime bins defined in (A). (Left) Combined expression of outer bulge marker genes is shown. Black line denotes median expression, with the 25th and 75th percentile colored in blue. (Right) Violin plots show the expression of selected outer bulge marker genes, ordered by pseudotime bins. (C) Expression of IFE marker genes (IFE > OB in telogen dataset with 99.9% PP) in Lgr5TOM cells, ordered by pseudotime bins defined in (A). (Left) Combined expression of IFE marker genes is shown. Black line denotes median expression, with the 25th and 75th percentile colored in green. (Right) Violin plots show the expression of selected IFE marker genes, ordered by pseudotime bins. (B and C) Blue and green lines and shadings in violin plots mark the median expression and 99% PP interval of telogen outer bulge (blue; data from Joost et al., 2016) and IFE cells (green; data from Joost et al., 2016). (D) Immunostainings of Lgr5-EGFP and CD34 in Lgr5TOM bulge and Lgr5TOM wound cells (8 days PWI). (E) Immunostaining of KRT6 in Lgr5TOM bulge and Lgr5TOM wound cells (1 day PWI). (D and E) Dotted lines: approximate location of the basement membrane (thin) and wound front (thick). Filled lines: approximate location of the skin surface. HG, hair germ; HS, hair shaft; SG, sebaceous gland. Scale bars: 100 μm (panoramas); 25 μm (zoom-ins). Note that tyramide signal amplification (TSA)-based antibody staining yields unspecific signal in the dermis and the sebaceous gland. See also Figure S4 and Table S3. Cell Reports 2018 25, 585-597.e7DOI: (10.1016/j.celrep.2018.09.059) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 Wound-Induced Remodeling of Cellular Interaction Potential with Wound Environment in Lgr5TOM Cells (A) Overview of the analytical approach: expression of receptors and ligands in the single-cell dataset (control and state 1) was compared to bulk RNA-seq data covering both the dermal and epidermal wound environment 1 day PWI. Receptors and ligands upregulated in the wound environment were identified, and a receptor-ligand database (Ramilowski et al., 2015) was used to identify receptor-ligand pairs between Lgr5TOM and Lgr6TOM wound cells and the wound environment (STAR Methods). (B) Bi-graph of potential receptor-ligand interactions between wound environment (1 day PWI) and Lgr5TOM and Lgr6TOM control and early wound cells (state 1). Arrow widths are proportional to the total number of potential receptor-ligand pairs between two populations and indicate the directionality of interaction (origin of arrow = ligand-expressing population). Arrow labels denote the number of receptor-ligand pairs (bold) and the probability of observing as many interactions at random. See also Figure S5 and Table S4. Cell Reports 2018 25, 585-597.e7DOI: (10.1016/j.celrep.2018.09.059) Copyright © 2018 The Author(s) Terms and Conditions

Figure 5 Identification of Key Ligands and Receptors Involved in Interaction between Wound Epidermis and Wound Environment at 1 Day PWI (A) Sankay plots showing potential interactions between ligands induced in the wound environment and receptors found in both Lgr5TOM and Lgr6TOM wound cells (upper panel) or between receptors induced in the wound environment and ligands found in both Lgr5TOM and Lgr6TOM wound cells (lower panel). (B) Expression of selected receptors and ligands in Lgr5TOM and Lgr6TOM control and early wound cells (shown is the expression of the state 1 subpopulation with the highest expression level) or in bulk-cell control and wound samples (1 day PWI). Expression values for single-cell data: log2-transformed PP distribution from negative binomial regression of molecule counts; crosses denote the median expression. Expression values for bulk cell data: mean expression. (C) Detection of Itgb1, Cd44, and Thbs1 by mRNA fluorescence in situ hybridization (RNA-FISH) in 1 day PWI sections. Dotted lines: approximate location of the basement membrane (thin) and wound front (thick). Filled lines: approximate location of the skin surface. WF, wound front. WGA, wheat germ agglutinin (stains cell membranes). Scale bars: 100 μm (panoramas); 50 μm (zoom-ins). See also Figure S5 and Table S4. Cell Reports 2018 25, 585-597.e7DOI: (10.1016/j.celrep.2018.09.059) Copyright © 2018 The Author(s) Terms and Conditions

Figure 6 Shared and Unique Transcriptional Signatures in Early Lgr5TOM and Lgr6TOM Wound Cells (A) Functional signatures most significantly enriched (q-value ≤ 0.05) in Lgr5TOM or Lgr6TOM wound cell states. Considered in the analysis were all genes induced with more than 99.9% PP. Molecular signatures were compiled from KEGG (Kyoto Encyclopedia of Genes and Genomes), Reactome, and MSigDB Hallmark databases as well as signatures linked to telogen populations in Joost et al. (2016). Shown is the relative −log10 q-value over all Lgr5TOM or Lgr6TOM states. (B) Comparison of Lgr5TOM and Lgr6TOM early wound cells (state 1; subpopulation with highest expression shown). Shown is the expression (log2-transformed PP distribution from negative binomial regression of molecule counts) of genes upregulated in both Lgr5TOM (blue violins) and Lgr6TOM (orange violins) wound cells or uniquely induced in only one of them. To control for differences in baseline expression (gray violins), a gene was only considered uniquely induced if it exceeded its matched control population and the highest expressing population of wound cells in the other background with 99.9% PP. (C) Immunostaining of STFA1 (CSTA) expression in Lgr6TOM and Lgr5TOM mice 1 day PWI. Dotted lines: approximate location of the basement membrane (thin) and wound front (thick). Filled lines: approximate location of the skin surface. Scale bars: 100 μm (panoramas); 25 μm (zoom-ins). Note that TSA-based antibody staining yields unspecific signal in the dermis and the sebaceous gland. See also Figure S6 and Table S5. Cell Reports 2018 25, 585-597.e7DOI: (10.1016/j.celrep.2018.09.059) Copyright © 2018 The Author(s) Terms and Conditions