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. 2020 Jun 12;10(17):7581-7598.
doi: 10.7150/thno.44306. eCollection 2020.

Single-cell Transcriptome Profiling reveals Dermal and Epithelial cell fate decisions during Embryonic Hair Follicle Development

Wei Ge  1 , Shao-Jing Tan  2 , Shan-He Wang  1 , Lan Li  2 , Xiao-Feng Sun  2 , Wei Shen  2 , Xin Wang  1
Affiliations

Single-cell Transcriptome Profiling reveals Dermal and Epithelial cell fate decisions during Embryonic Hair Follicle Development

Wei Ge et al. Theranostics. .

Abstract

It is estimated that 50% of men and 25% of women worldwide suffer from hair loss, and therefore it is of great significance to investigate the molecular pathways driving hair follicle de novo morphogenesis. However, due to high cellular heterogeneity and the asynchronous development of hair follicles, our current understanding of the molecular mechanisms involved in follicle development remains limited. Methods: Single-cell suspensions from the dorsal skin of E13.5 (induction stage), E16.5 (organogenesis) fetal mice, and newborn mice (cytodifferentiation stage, postnatal day 0, P0) were prepared for unbiased single-cell RNA sequencing. To delineate the single-cell transcriptional landscape during hair follicle de novo morphogenesis, we performed t-distributed Stochastic Neighbor Embedding (tSNE), pseudotime cell trajectory inference, and regulon enrichment analysis to dissect cellular heterogeneity and reveal the molecular pathways underlying major cell type cell fate decisions. To validate our analysis, we further performed immunohistochemistry analysis of the key molecules involved during hair follicle morphogenesis. Meanwhile, intercellular communication between different cell populations was inferred based on a priori knowledge of ligand-receptor pairs. Results: Based on tSNE analysis, we identified 14 cell clusters from skin tissue and delineated their cellular identity from specific gene expression profiles. By using pseudotime ordering analysis, we successfully constructed the epithelium/dermal cell lineage differentiation trajectory. For dermal cell lineage, our analysis here recapitulated the dynamic gene expression profiles during dermal condensate (DC) cell fate commitment and delineated the heterogeneity of the different dermal papilla (DP) cell populations during in utero hair follicle development. For the epithelium cell lineage, our analysis revealed the dynamic gene expression profiles of the underappreciated matrix, interfollicular epidermis (IFE), hair shaft and inner root sheath (IRS) cell populations. Furthermore, single-cell regulatory network inference and clustering analysis revealed key regulons during cell fate decisions. Finally, intercellular communication analysis demonstrated that strong intercellular communication was involved during early hair follicle development. Conclusions: Our findings here provide a molecular landscape during hair follicle epithelium/dermal cell lineage fate decisions, and recapitulate the sequential activation of core regulatory transcriptional factors (TFs) in different cell populations during hair follicle morphogenesis. More importantly, our study here represents a valuable resource for understanding the molecular pathways involved during hair follicle de novo morphogenesis, which will have implications for future hair loss treatments.

Keywords: Cell fate decision; Hair follicle morphogenesis; Single-cell transcriptome.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Overview of experimental procedures and characterization of major cell populations from embryonic skin tissues. (A) Schematic diagram illustrating the experimental pipeline for scRNA-seq analysis of embryonic skin tissues. Single-cell transcriptomes were obtained based on the 10x Chromium platform. (B) tSNE plot of embryonic single skin cells. Each point represents one single cell and cells in the same cluster represents high similarity in transcriptome profile. The left plot depicts tSNE plot of the integrated dataset from 3 different time point and cells were color-coded with developmental time point. The right plot depicts 14 transcriptional distinct cell clusters and cells were color-coded with cluster information. (C) Visualization of dermal and epithelial marker gene expression across all single cells in the tSNE plot. Col1a1 and Lum were used to mark dermal cell populations and Krt14 and Krt15 were used to mark epithelium populations. (D) Characterization of major cell types in the embryonic skin tissues in the tSNE plot. Cells were labeled with their cell identity and were color-coded. (E) Dot plot depicts representative dermal cell, epithelial, melanocyte, and pericyte gene expression. The dot size represents the percentage of cells expressed and the color intensity represents relative expression level.
Figure 2
Figure 2
Recapitulating dermal cell fate decision towards DC fate. (A) Diagram deciphering dermal lineage and epidermal lineage differentiation at E13.5 to E16.5. (B) Pseudotime ordering of all dermal cell lineage cells. Each dot represents one cell and each branch represents one cell state. The left plot was labeled with developmental time and the right plot was labeled with cell states. (C) Heatmap illustrating the (differentially expressed genes, DEGs) dynamics towards DC and fibroblast fate along pseudotime. The DEGs were clustered into 4 gene sets according to k-means and the expression curve was illustrated in the middle. GO terms enriched for each gene set were labeled in the right panel. DC fated represents cell fate 2 in Figure 2B, while dermal adipocyte progenitors represent cell fate 1 in Figure 2B. (D) Immunofluorescence analysis of PRDM1, SOX9, LEF1, PCNA, CTNNB1, KRT15 and BMP2 expression in the E16.5 dorsal skin. Scale bars, 50 μm. (E) SCENIC binary regulon activity heatmap depicting DC and fibroblast enriched regulons. The column depicts a single cell while the row depicts regulons. For the regulons of particular interest, their representative binding motif was visualized in the right panel. “On” depicts active, while “Off” represents inactive. (F) Sequential visualization of enriched regulon activity in each gene set corresponding to Figure 2C. Their representative target genes were also provided and the red ticks depict confirmed markers in DC fate commitment.
Figure 3
Figure 3
Dissecting DP population heterogeneity in the late stage of hair follicle development. (A) Monocle pseudotime trajectory construction analysis and immunohistochemistry analysis of SOX2 expression in P0 skin. Arrows indicate the SOX2 positive DP population. Scale bars, 25 µm. (B) Heatmap illustrating DP signature gene dynamics from pre branch to ZZ DP and G/AA-DP fate. The corresponding GO terms for each gene set were listed in the right panel. (C) ZZ DP makers Bmp4, Sox18 expression and G/AA-DP markers Fgf7, Vcan expression along pseudotime. Cells were color-coded with cell states and the solid line represents cell fate 1, while the dashed line represents cell fate 2. (D) Comparison of DP signature genes in this study with previously identified different DP signature genes using bulk RNA seq. No. of genes depicts the number of overlapped genes. (E) Heatmap comparasion of enriched GO terms among 4 different gene sets. (F) SCENIC binary regulon activity heatmap deciphering G/AA-DP and ZZ DP branch specific enriched regulons. “On” depicts active, while “Off” represents inactive.
Figure 4
Figure 4
Dissecting molecular signature underlying matrix and IFE precursor fate commitment. (A) Histology analysis of E16.5 embryonic skin. Scale bar, 50 µm. (B) Pseudotime ordering of all epithelium cell populations from three developmental time stages. Each dot represents one cell. The left plot was color-coded with stage information, while the right plot was color-coded with developmental stages. (C) Heatmap displaying branch specific DEGs expression in branch point 1 in Figure 4B. Cell fate 1 indicates matrix fate and cell fate 2 indicates IFE fate. The corresponding expression curve and enriched GO terms for each gene set were visualized in the middle panel. The representative DEGs for each gene set were shown in the rectangular box (right panel) with red depicting confirmed signature genes. (D) Expression dynamics of representative branch specific marker genes along pseudotime. (E) Immunofluorescence analysis of CTNNB1, PRDM1, KRT15, BMP2, SOX9 and VDR expression in E16.5 skin tissues. Scale bar, 50 µm. (F) Binary regulon activity heatmap illustrating the branch-specific enrichment of key regulons. The representative regulons and their corresponding targets (500 bp upstream of TSS) were listed in the middle panel. The binding motif was listed in the right panel.
Figure 5
Figure 5
Dissecting hair shaft and IRS fate commitment from matrix precursors. (A) Histology analysis of P0 mouse skin. Scale bar, 50 µm. (B) Pseudotime visualization of the hair shaft and IRS fate decisions in the epithelium single cell pseudotime trajectory. (C) Heatmap illustrating branch specific DEGs dynamics along pseudotime. Cell fate 1 indicates hair shaft fate and cell fate 2 indicates IRS fate. (D) Binary regulon activity heatmap demonstrating branch specific enrichment of regulons. The cell states correspond to Figure 5 B. The representative regulon and binding motifs were listed in the right panel. (E) DEGs expression dynamics and GO enrichment in each gene set. The gene expression curve was listed in the left panel and GO terms for each gene set were listed in the right panel. (F) Immunofluorescence analysis of BMP2, CTNNB1, LEF1, SOX9, KRT15, PCNA, and BMP15 in P0 skin. Scale bar, 25 µm.
Figure 6
Figure 6
Intercellular ligand-receptor prediction. Ligand-receptor pairs between E13.5-E16.5 (A) and E16.5-P0 (B) main cell populations. Different cell populations were color-coded and ligand-receptor pairs were linked with a solid line.
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References

    1. Vary JC Jr. Selected Disorders of Skin Appendages-Acne, Alopecia, Hyperhidrosis. Med Clin North Am. 2015;99:1195–211. - PubMed
    1. Chen Y, Huang J, Chen R, Yang L, Wang J, Liu B. et al. Sustained release of dermal papilla-derived extracellular vesicles from injectable microgel promotes hair growth. Theranostics. 2020;10:1454–78. - PMC - PubMed
    1. Kageyama T, Yoshimura C, Myasnikova D, Kataoka K, Nittami T, Maruo S. et al. Spontaneous hair follicle germ (HFG) formation in vitro, enabling the large-scale production of HFGs for regenerative medicine. Biomaterials. 2018;154:291–300. - PubMed
    1. Paus R, Muller-Rover S, Van Der Veen C, Maurer M, Eichmuller S, Ling G. et al. A comprehensive guide for the recognition and classification of distinct stages of hair follicle morphogenesis. J Invest Dermatol. 1999;113:523–32. - PubMed
    1. Schmidt-Ullrich R, Paus R. Molecular principles of hair follicle induction and morphogenesis. Bioessays. 2005;27:247–61. - PubMed

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