Embryonic attenuated Wnt/β-catenin signaling defines niche location and long-term stem cell fate in hair follicle

Abstract

Long-term adult stem cells sustain tissue regeneration throughout the lifetime of an organism. They were hypothesized to originate from embryonic progenitor cells that acquire long-term self-renewal ability and multipotency at the end of organogenesis. The process through which this is achieved often remains unclear. Here, we discovered that long-term hair follicle stem cells arise from embryonic progenitor cells occupying a niche location that is defined by attenuated Wnt/β-catenin signaling. Hair follicle initiation is marked by placode formation, which depends on the activation of Wnt/β-catenin signaling. Soon afterwards, a region with attenuated Wnt/β-catenin signaling emerges in the upper follicle. Embryonic progenitor cells residing in this region gain expression of adult stem cell markers and become definitive long-term hair follicle stem cells at the end of organogenesis. Attenuation of Wnt/β-catenin signaling is a prerequisite for hair follicle stem cell specification because it suppresses Sox9, which is required for stem cell formation.

Keywords: Wnt; developmental biology; hair follicle; mouse; niche; regeneration; self-renewnal; stem cell; stem cells.

Figure 1.. Embryonic cellular origin of adult hair follicle stem cells.

(A) Diagram of hair follicle morphogenesis and the lineage-tracing experiment. All lineage-tracing experiments ended at the first telogen, but started at different stages including the placode, hair germ, and hair peg stages. (B,C) Representative images of tail skin hair follicle organogenesis. Red boxes indicate the regions used for quantification in the lineage-tracing experiments. The hair cycle in tail skin progresses along the anterior to posterior and in the dorsal to ventral directions. At postnatal day 1 (P1), in the chosen region, the primary central hair follicles are in the hair peg stage while the secondary outer follicles are in the placode stage. At P15, in the chosen region, primary central hair follicles are in the telogen phase. Scale bar: 1500 μm for the whole mount image; 100 μm for the enlarged images. (D) Summary of the lineage-tracing experiments with: Shh-CreER::Rosa-stop-mTmG, Nfatc1-CreER::Rosa-stop-mTmG, and Lgr5-GFP-CreER::Rosa-stop-tdTomato::K14H2BGFP mice. Percentages of labelled HFSCs at the telogen phase were quantified using whole mount sample images. Representative images are single confocal Z slices from the data used for quantification. N=5 mice, >200 HFs. Raw data are plotted to the left of each box-and-whisker plot: the median and the 25th and the 75th percentiles are denoted by notches and the bottom and top boxes, respectively; the 5th and 95th percentiles are denoted as whiskers. Scale bar: 50 μm. (E) In both the hair germ and the hair peg, the progenies of Shh+ cells in the center of HFs express the differentiation marker Keratine 6 (Krt6). Scale bar: 50 μm. (F) Lineage-tracing experiment starting at the hair peg stage and ending at morphogenesis anagen (growth phase) using Nfatc1-CreER::Rosa-stop-mTmG mice. Note that the labeled cells stay dormant at their original position without contributing to HF down growth. Scale bar: 50 μm. (G) Model depicting the spatial and temporal pattern of hair follicle stem cell emergence. DOI: http://dx.doi.org/10.7554/eLife.10567.003

Figure 1—figure supplement 1.. Difference between back and tail skin hair follicle organogenesis.

(A) In mouse back skin, hair follicle initiation occurs via three separate waves that start, respectively, around E13.5, 15.5, and 18.5. At any given embryonic time point between E13.5 to E18.5, there is a mixture of hair follicles at different developmental stages in back skin, lacking any discernable pattern. After birth, the three separate waves synchronize into similar anagen and then telogen. Thus, it is impossible to pinpoint the exact early developmental stage of any telogen back skin hair follicle at the beginning of the lineage-tracing experiments when Tamoxifen was injected at an embryonic time point. (B) Tail skin hair follicles are arranged in triplets, and the growth of two secondary outer follicles is typically initiated next to a primary central follicle after it has already begun development. Within the dorsal middle one-third section of tail skin, at P1, the primary central hair follicles are at the hair peg stage and the secondary outer follicles on the side are at the placode stage. At P6, all three hair follicles in a triplet progress to anagen. At P15, the primary central hair follicles have already entered telogen or even the telogen to anagen transition stage, while the secondary outer hair follicles on the side are still in the catagen to telogen transition. At P21, the primary central hair follicles are in full anagen and the two secondary outer follicles are at the telogen to anagen transition stage. Scale bars: 50 µm. DOI: http://dx.doi.org/10.7554/eLife.10567.004

Figure 1—figure supplement 2.. Diagram, quantification, and specificity of CreER lines for the lineage-tracing experiments.

(A) Diagram of the lineage-tracing experiment using Nfatc1-CreER::Rosa-stop-mTmG mice. (B) Diagram of the lineage-tracing experiment using Lgr5-GFP-CreER::Rosa-stop-tdTomato::K14H2BGFP mice. Representative images used here are the same representative images shown in Figure 1D to indicate these diagram are drawn to explain the experimental designs for Figure 1D. Scale bar: 50 µm. (C) Diagram depicting the individual time points used for the lineage-tracing experiments starting from different early developmental stages. Green color highlights the target hair follicles used in the designated lineage-tracing experiments. (D–F) Percentage of HFs with labeled HFSCs at telogen. Tracings starting from the placode stage (D), the hair germ stage (E), and the hair peg stage (F). (G) Nfatc1-CreER::Rosa-stop-mTmG, Shh-CreER::Rosa-stop-mTmG, Lgr5-GFP-CreER::Rosa-stop-tdTomato::K14-H2BGFP mice tail skin hair follicles with or without Tamoxifen induction. Scale bar: 200 µm. DOI: http://dx.doi.org/10.7554/eLife.10567.005

Figure 2.. Niche position in the hair peg determines hair follicle stem cell fate.

(A) Diagram of two-photon-mediated cell ablation experiment using Nfatc1-CreER::Rosa-stop-tdTomator::K14H2BGFP mice. (B) Representative images of hair follicles before and after cell ablation in a live animal. (C) Representative whole-mount images of hair follicles 15 days after cell ablation. Notice that the control hair follicle has tdTomato+ cells in the bulge while the ablated hair follicle has a normal bulge composed of tdTomato- cells. (D) Quantification of hair follicle stem cell number at telogen using whole mount samples. N=6 mice, >12 HFs. (E) Quantification of percentage of tdTomato+ outer bulge cells at telogen using whole mount samples. N=6 mice, >12 HFs. (F–H) Representative images of hair follicles before (F), immediately after (G), and 21 days after (H) cell ablation from the same mouse. Notice that both the control hair follicle and ablated hair follicle enter anagen. (I) Quantification of hair follicles that have started regeneration 21 days post cell ablation. N=5. Scale bars: 50 μm DOI: http://dx.doi.org/10.7554/eLife.10567.006

Figure 2—figure supplement 1.. Ablation specificity, Tamoxifen induction efficiency, and whole-mount views of the two-photon-mediated cell ablation experiments.

(A) Representative images of hair follicles before, immediately after, and 2 days post ablation in the same live mouse. Notice the ablated hair placodes disappear, while the follicles adjacent to them develop normally. Scale bar: 50 µm. (B) Whole-mount tail skin image of Nfatc1-CreER::Rosa-stop-mTmG mice with lineage tracing starting at the hair peg stage. The percentage of labeled HFSCs decreased along the dorsal-ventral axis starting from the dorsal anterior-posterior midline. Scale bar: 500 µm. (C) Whole-mount image of tail skin used for quantification 21 days after ablation. Ablated hair follicles are numbered and highlighted in red. Scale bar: 500 µm. DOI: http://dx.doi.org/10.7554/eLife.10567.007

Figure 3.. Unbiased RNA-seq analysis reveals factors that define the hair peg niche.

(A) Diagram of the FACS experiments using Nfatc1- and Shh-CreER::Rosa-stop tdTomato::K14H2BGFP mice. (B) FACS isolation of distinct GFP+RFP+ populations to obtain Shh+ and Nfatc1+ epithelial cells. (C) Unsupervised hierarchical clustering and heat map display of genes that were differentially expressed between Shh+ cells and Nfatc1+ cells. N=2 (D) Gene Ontology analysis of ≥2-fold up-regulated genes in Shh+ cells compared to Nfatc1+ cells. The Wnt signaling pathway is highlighted. (E) Validation of differentially expressed genes using qPCR. N=3. (F–G) In situ staining of Axin2 (F) and Wnt10b (G) in developing hair follicles. (H) Shh+ cells are Wnt/β-catenin signal responsive cells. Shh expression was represented by tdTomato in Shh-CreER::Rosa-stop-tdTomato mice. Wnt/β-catenin-responsive cells were detected by β-gal staining in TOPGAL mice. Scale bars: 50 μm. DOI: http://dx.doi.org/10.7554/eLife.10567.008

Figure 3—figure supplement 1.. RNA seq results of representative genes from different populations.

(A) RNA seq results of Nfact1 and Shh from different populations. N=2 (B) Fold enrichment of canonical Wnt pathway genes from the RNA-seq results. N=2. DOI: http://dx.doi.org/10.7554/eLife.10567.010

Figure 4.. Attenuated Wnt/β-catenin signaling is uniquely associated with hair follicle stem cell specification.

(A–B) Lineage tracing with Axin2CreER::Rosa-stop-mTmG mice. N=5 mice, >120 HFs. (C) Descendants of Gli1+ cells were represented by the expression of tdTomato in Gli1-CreER::Rosa-stop-tdTomato::K14H2BGFP mice. Note that Gli1+ descendants are located in almost all the hair follicle cells at the placode, hair germ, and hair peg stages. (D) Descendants of Id2+ cells were represented by the expression of tdTomato in Id2-CreER::Rosa-stop-tdTomato::K14H2BGFP mice. Note that Id2+ descendants were only located at the hair bulb stage matrix and pre-cortex area. (E) Diagram summarizing the descendants of the Wnt-, Shh-, and BMP-signal responsive cells at different early developmental stages. Scale bars: 50 μm. DOI: http://dx.doi.org/10.7554/eLife.10567.011

Figure 5.. Elevated Wnt/β-catenin signaling abolishes hair follicle stem cell specification and suppresses Sox9 expression in hair follicles.

(A) Diagram of the experiments using Nfatc1-CreER::Rosa-stop-tdTomato::Exon3-Ctnnb1^fl/wt mice. (B) Nuclear β-catenin staining indicates successful activation of Wnt/β-catenin signaling in upper hair follicle. (C–E) Abolished bulge niche formation and hair follicle stem cell specification in exon3-Ctnnb1 Het HFs compared to WT HFs. Krt6 (C) is a marker for inner layer bulge cells serving as a niche that can maintain quiescence for outer layer HFSCs. Sox9 (D) and CD34 (E) are adult hair follicle stem cell markers. (F) Wnt/β-catenin signal responsive cells, represented by β-gal positive cells in TOPGAL mice, do not express Sox9 during normal development. (G) Activation of Wnt/β-catenin signaling suppresses Sox9 expression in vivo. The arrows point to protrusions resulting from elevated Wnt/β-catenin signaling in exon3-Ctnnb1 Het HFs. (H) Diagram of the experiments using K14-rtTA::teto-Cre::Ctnnb1^fl/fl mice. β-catenin is conditionally deleted in epithelial cells by feeding mice with Doxycyclin from E17.5 to P11. Samples were taken at P1 and P11. (I–J) Loss of Wnt/β-catenin signaling leads to expanded Sox9 expression in HFs at both the hair germ stage (I) and in postnatal skin (J). Scale bars: 50 μm. DOI: http://dx.doi.org/10.7554/eLife.10567.012

Figure 5—figure supplement 1.. Effects of both gain and loss of function studies targeting Wnt/β-catenin signaling in skin.

(A) Hematoxylin and eosin (H&E) staining of exon3-Ctnnb1 Het and control tail skin sections from P15 to P55. Control hair follicles enter into the first anagen from P15 to P25. Exon3-Ctnnb1 Het follicles enter into anagen around P45. Afterwards they return to telogen at P55, and new bulge structures reform. Scale bar: 50 µm. (B) Tail skin hair coats of control and exon3-Ctnnb1 Het mice at different time points. Notice in Het mice the disappearance of the hair coat at P30 and its reappearance at P100. Scale bar: 1 mm. (C) De novo formation of a normal niche in exon3-Ctnnb1 Het mice at P100. Note the recovered expression of the stem cell markers CD34 and Sox9; scale bar: 50 µm. (D) Spontaneous disappearance of exon3-Ctnnb1 Het-expressing cells shown by genotyping of isolated cells at different time points. (E) Conditional ablation of epithelium β-catenin leads to hair follicle development defects. β-catenin can be detected in the control mice but not in the epithelia of knockout mice. Scale bar: 50 µm. (F) Hair coat differences between WT and β-catenin cKO mice at P11. DOI: http://dx.doi.org/10.7554/eLife.10567.013

Figure 6.. Embryonic Wnt/β-catenin signaling diminishes the long-term self-renewal ability of hair follicle stem cells in adult.

(A) Diagram depicting the time points of the lineage tracing experiment. (B–C) Long-term lineage tracing experiments using Axin2-CreER::Rosa-stop-mTmG mice. Note the disappearance of labelled HFSCs in the third telogen. Representative images shown are single confocal Z slice from the data used for quantification. N=3 mice, >110 HFs. ***p<0.0001. (D) Long-term lineage tracing experiments with Sox9-CreER::Rosa-stop-mTmG mice. Arrowheads indicate ORS and arrows indicate terminally differentiated layers. Note the change in downstream progeny fates of labeled cells at different hair cycles. Scale bars: 50 μm. DOI: http://dx.doi.org/10.7554/eLife.10567.014

Figure 6—figure supplement 1.. Long term cell fate of Lgr5-CreER labelled hair peg cells.

(A) Diagram depicting the time points of the lineage tracing experiment. (B–C) Long-term lineage tracing experiments using Lgr5-CreER::Rosa-stop-tdTomato mice. Note the persistence of a small percentage of labelled HFSCs up to the ~6^th telogen. Representative images shown are single confocal Z slice from the data used for quantification. N=3 mice, >100 HFs. Scale bars: 50 μm. DOI: http://dx.doi.org/10.7554/eLife.10567.015

Figure 7.. Model illustrating that long-term hair follicle stem cell emergence results from progenitors occupying an embryonic niche location, which is defined by the absence of Wnt/β-catenin signaling that would otherwise block the expression of a key factor required for stem cell specification.

DOI: http://dx.doi.org/10.7554/eLife.10567.016