Epithelial-Mesenchymal Micro-niches Govern Stem Cell Lineage Choices

Abstract

Adult tissue stem cells (SCs) reside in niches, which, through intercellular contacts and signaling, influence SC behavior. Once activated, SCs typically give rise to short-lived transit-amplifying cells (TACs), which then progress to differentiate into their lineages. Here, using single-cell RNA-seq, we unearth unexpected heterogeneity among SCs and TACs of hair follicles. We trace the roots of this heterogeneity to micro-niches along epithelial-mesenchymal interfaces, where progenitors display molecular signatures reflective of spatially distinct local signals and intercellular interactions. Using lineage tracing, temporal single-cell analyses, and chromatin landscaping, we show that SC plasticity becomes restricted in a sequentially and spatially choreographed program, culminating in seven spatially arranged unilineage progenitors within TACs of mature follicles. By compartmentalizing SCs into micro-niches, tissues gain precise control over morphogenesis and regeneration: some progenitors specify lineages immediately, whereas others retain potency, preserving self-renewing features established early while progressively restricting lineages as they experience dynamic changes in microenvironment.

Keywords: epithelial-mesenchymal interactions; lineage determination; single-cell analyses; stem cells; tissue regeneration.

Figure 1. The Eight Concentric Layers of the Mature HF

(A) Schematic depicting resting (Telogen) and regenerating (Anagen) phases of hair follicles. Bu-HFSC, HF stem cells residing in the bulge niche; HG, hair germ, a niche housing the ‘primed’ HFSCs that will be activated at the start of the new hair cycle; DP; dermal papilla, specialized mesenchymal cells required to fuel the hair cycle. (B) (left), Schematic depicting the complex organization of the mature HF. TACs, transit amplifying cells, which in full anagen balance proliferation and differentiation to produce the hair shaft (HS) and its channel, the inner root sheath (IRS). ORS, outer root sheath; Cp, companion layer; He, Henle’s layer; Hu, Huxley’s layer; Ci, IRS cuticle; Ch, HS cuticle; Co, cortex; Me, medulla. (right), Ultrastructure of sagittal section just above the mature hair bulb. Scale bar = 10μm. (C) Lineage heterogeneity revealed by immunofluorescence for: GATA3 (Hu, Ci); K71 (He); K6 (Me, Cp); K32 (Ch); K31 (Co). Scale bars=50μm.

Figure 2. Single Cell RNA-seq Analysis Reveals Molecular Heterogeneity of TACs

(A) Experimental strategy to purify the TACs at the epithelial-mesenchymal border. (left), Expression patterns of P-cadherin, Integrin β1 and transgenic Lhx2-GFP in the hair bulb. (right), Purification scheme. (B) Scatter plots showing correlation between (left) two batches of single cell RNA-seq and (right) two biological replicates of RNA-seq on bulk FACS-sorted populations. (C) (left), Unbiased clustering of transcriptomes of individual basal TACs from Ana-VI HFs. Each cell is represented as a dot, colored by a clustering algorithm and plotted on the tSNE graph. (right), Heatmap showing transcriptome similarities between single cells measured by Pearson’s correlation coefficient matrix. Clusters are color-coded along the axes. (D) (top) tSNE plot showing the cell cycle status of each cluster. (bottom), tSNE plot showing Tchh expression. Although a marker of differentiating medulla and IRS cells, trichohyalin (Tchh) was only expressed in basal medulla progenitors (arrows). The dotted line denotes epidermal-dermal border. Note that Cluster 1 (C1) is non-proliferative and expresses many medulla genes. (E) Immunofluorescence showing EdU⁺ S-phase cells, Integrin α6 and P-cadherin in the mature hair bulb. (F) tSNE plots showing 2^nd-level subclustering of C2 and C3 into six (left) and two (right) subclusters, respectively. Scale bars = 50μm. See also Figures S1 and S2; Tables S1 and S2.

Figure 3. Assigning the Identities of Molecularly Distinct TAC Progenitors

(A) Heatmap showing relative expression levels of signature genes for each cluster. Cells were ordered by clusters. (B) (left), Gene ontology analyses of subclusters displaying enriched IRS and HS genes (>2X). (right), tSNE plots show that Cluster C2 is composed of HS (C2-a,b,c) and IRS (C2-d,e,f) subclusters, which were identified according to transcriptome. Genes in red are regulated by TAC ‘super-enhancers’, reflective of key lineage identity genes (Adam et al., 2015). (C) (left), tSNE plot showing high Gata3 expression in C2-d and C2-e IRS populations. (right), Immunofluorescence confirms the identity of these sub-clusters. (D) (left), tSNE plot showing high Prdm1 expression in C2-a, C2-b, C2-c and C2-f. (right), Immunofluorescence confirms the identity of these sub-clusters. (E) tSNE plot showing two sub-clusters of C3, which represent companion layer (CP, Krt75⁺) and lower proximal cup (LPC, Lgr5⁺). (F) Table summarizing certain genes diagnostic for assigning identities to proliferative basal TAC subclusters. In all, each subcluster displayed a signature of ~100 genes up by ≥2X relative to other subclusters. Scale bars = 50μm. See Table S3 for full signature lists.

Figure 4. TACs Consist of Spatially Arranged Uni-Lineage Progenitors That Divide Asymmetrically Relative to the Basement Membrane

(A) (left), Lineage tracing strategy with R26-Confetti reporter mouse and lentivirus expressing CreER driven by pSMAD2-binding elements (SBE, TGFβ-responsive element), active only at late Telo-AnaII. (middle), In Ana-II, pSMAD2 labels progenitors adjacent to the DP. (right), Lineage-tracing marks cellular columns of uni-lineage cells. Scale bars = 50μm. (B) Ultrastructure of mature hair bulb. TACs, orange; pre-medulla cells, purple; mitotic cells, green. (C) Basal TACs divide perpendicularly to the basement membrane. Late anaphase daughters are marked by SURVIVIN (example shown), allowing quantification of division angles relative to the underlying basement membrane. Scale bars = 10μm. (D) Schematic depicting basal TACs as unipotent progenitors. See also Figure S3.

Figure 5. Lineage Restriction Occurs in a Temporally Sequential Manner

(A) Lineage-tracing (as in Figure 4) also gave rise to adjacent lineages marked by same fluor, suggestive of multi-lineage progenitors born at an earlier stage in the regenerative phase. (B) Single cell analysis of Ana-II TACs reveals multi-lineage progenitors with features of both IRS and HS, as well as unipotent progenitors of the lower proximal cup (LPC) and companion layer (CP). (C) Heatmap shows relative expression levels of signature genes for each cluster. Cells are ordered by clusters. (D) (top), tSNE plots show co-expression of IRS lineage marker (Nrp2) and HS lineage marker (Hoxc13) in multipotent progenitors. (bottom), Multipotent progenitors are enriched for key IRS and HS lineage progression genes. Genes in red are enriched in Ana-VI IRS TACs, genes in blue are enriched in Ana-VI HS TACs and genes in black are enriched in both lineages. (E) Dual expression of key IRS (GATA3) and HS (HOXC13) lineage TFs in a subset of Ana-II TACs. Note that by Ana-III, these TFs have become restricted to their different lineages. (F) (top), tSNE plots showing high Krt79 and Gata6 expression in companion (CP) progenitor population. (bottom), Some representative genes enriched in CP cluster (>2-fold). (right), GATA6 immunostaining illustrates the emergence of CP unipotent progenitors (arrows) at Ana-II. (I) Venn Diagrams show similarity overlaps between Ana-II and Ana-VI progenitors. Scale bars = 50μm. See also Figures S4 and S5 and Table S4.

Figure 6. Lineage Diversity Begins During Quiescence

(A) (left), Ultrastructure of HF at telogen and Ana-I. Bulge-HFSCs in green; hair germ (HG) in red; DP in blue. Note perpendicular anaphase mitosis in yellow. Scale bars = 25μm. (B) tSNE plot showing that unbiased clustering of telogen HFSCs, telogen HG cells and Ana-I HG cells identifies 6 distinct progenitor states. Each cell is represented as a dot and colored by a clustering algorithm. (C) Heatmap shows relative expression levels of signature genes for each cluster. Cells are ordered by clusters. (D) (left) Table shows a list of representative signature genes for each cluster. (right) Marker-based identification (tSNE plots) of HFSC and HG clusters. Shown are expression patterns of 6 representative signature transcripts. (E) The switch between super-enhancer regulated HFSC and TAC identity genes occurs precociously in Telo-HG3, revealing a strong regional bias within primed basal SCs of the telogen HG. (F) Chromatin accessibility (profiled by ATAC-seq) of the genes encoding the 6 signature transcripts from (D) and assayed in HG versus bulge SCs in mid-telogen. Note that the four representative signature genes of the bulge HFSC cluster have reduced chromatin accessibility in HG, while the two representative signature genes of HG clusters have lower accessibility in bulge. See also Figure S6 and Tables S5.

Figure 7. Differential BMP and WNT Signaling Establishes Compartmentalization of Epithelial-Mesenchymal Micro-niches

(A) (left), pSMAD1/5/9 immunofluorescence reveals heterogeneity within the telogen HG, reflected in corresponding repression of BMP target genes Id2 and Sbsn. (middle) pSMAD1/5/9 in Ana-I and Ana-III HF. (right), Genes regulated by BMP (Genander et al., 2014) correlate with SMAD1 activity in primed SC (HG) populations. (B) Gene set enrichment analysis showing similarities between Bmpr1a-null HFSCs and HG3 showing low BMP signaling. (C) (left), Immunofluorescence and single cell analyses reveals heterogeneity among quiescent HG cells in their suppression of SOX9/Sox9 concomitant with induction of LEF1/Lef1 induction. Note the appearance of LEF1⁺ DP cells opposite the Lef1^Hi Telo-HG3 cells (arrow), and the broadening of this zone with onset of regenerative phase. (D) Ana-VI DP was purified as LEF1-RFP⁺Integrinα9⁺K14-H2BGFP⁻ cells. (E) Heterogeneity in basal TACs is mirrored by heterogeneity in DP cells. (left), tSNE plot showing that unbiased clustering of FACS-purified Ana-VI DP cells identifies 4 subpopulations. Each cell is represented as a dot and colored by a clustering algorithm. (right) Heatmap shows relative expression levels of signature genes for each cluster. Cells are ordered by clusters. (F) In situ hybridization and immunofluorescence to spatially map Ana-VI DP subpopulations. (G) Patterns of WNT and BMP signaling and expression of downstream target genes in Ana-VI DP clusters. (H) Schematic depicting spatial organization of epithelial-mesenchymal cross-talk between opposing TAC and DP micro-niches in Ana-VI HF. Scale bars = 25μm. See also Figure S7 and Table S6.