BMP signaling and its pSMAD1/5 target genes differentially regulate hair follicle stem cell lineages

Abstract

Hair follicle stem cells (HFSCs) and their transit amplifying cell (TAC) progeny sense BMPs at defined stages of the hair cycle to control their proliferation and differentiation. Here, we exploit the distinct spatial and temporal localizations of these cells to selectively ablate BMP signaling in each compartment and examine its functional role. We find that BMP signaling is required for HFSC quiescence and to promote TAC differentiation along different lineages as the hair cycle progresses. We also combine in vivo genome-wide chromatin immunoprecipitation and deep-sequencing, transcriptional profiling, and loss-of-function genetics to define BMP-regulated genes. We show that some pSMAD1/5 targets, like Gata3, function specifically in TAC lineage-progression. Others, like Id1 and Id3, function in both HFSCs and TACs, but in distinct ways. Our study therefore illustrates the complex differential roles that a key signaling pathway can play in regulation of closely related stem/progenitor cells within the context of their overall niche.

Figure 1. BMP signaling is temporally regulated and required to maintain matrix TACs

(A) pSMAD1/5 patterns throughout the hair cycle. (B) Early anagen HFSCs fail to downregulate pSMAD1/5 after Shh ablation. (C) Bmpr1a ablation leads to precocious HFSC activation. (D) Quantifications of changes in %Ki67+ hair bulb cells with time following Bmpr1a ablation in HFSCs. (E) Long term loss of BMP signaling leads to HF cysts composed largely of ORS (K17+), early matrix (LEF1+) and IRS-TACs (GATA3+). (F) Within a planar midsection of the hair bulb, more central cortical HS-TACs exhibit stronger nuclear pSMAD1/5 reactivity than more distal GATA3+ TACs. Cre-promoters are given in parentheses. Ana, anagen; Telo, telogen; Bu, bulge; HG, hair germ; DP, dermal papilla; Mx, matrix; Cx, cortex; IRS, inner root sheath. Data are represented as mean±SEM. *** = p<0.001 using Anova. Scale bars = 25 μm.

Figure 2. BMP restricts the Shh-expressing IRS progenitor pool

(A) Experimental timeline for selectively targeting Bmpr1a ablation in Shh-expressing TACs during full-anagen. (B–D) BMPR1a loss results in an expansion of YFP⁺ cKO TACs and an increase in their proliferation. (E) Lineage tracing of YFP⁺ TAC progeny. (F and G) Differential distribution of YFP⁺ progeny reveals an increase in AE15⁺ IRS and decrease in AE13⁺ cortex following Bmpr1a ablation in TACs. (H) Bmpr1a-null YFP⁺ TACs lineage trace to AE15⁺ IRS but not AE13⁺ cortex. (I) Axin2-LacZ activity in Shh-expressing TACs, DP and cortex/medulla. Note lack of X-Gal staining (blue) in HS developing from Bmpr1a-targeted side. (J) LEF1+ TACs are expanded upon BMPR1a loss, but LEF1+ cortex is absent. (K) Shh⁺ IRS TACs are expanded and express elevated Shh in the absence of BMP signaling. (L) GATA3⁺ cells are expanded following Bmpr1a ablation in Shh⁺ TACs. (M and N) Lineage tracing of YFP⁺ progeny following Gata3 ablation in Shh⁺ TACs. Following loss of GATA3, AE15+ IRS progeny (external to the K82⁺ HS cuticle) are lost, while AE15+ HS progeny (internal to K82) are expanded. (O) Gata3 ablation in Shh⁺ TACs leads to an expanded cortical layer (AE13+) and smaller AE15+ IRS. (P) EM of Gata3 straight KO HFs reveals absence of IRS lineage. (Q) Proliferation is unaffected following Gata3 ablation in TACs. Data are represented as mean±SEM. * = p<0.05, ** = p<0.01, *** = p<0.001, Students t-test. Scale bar = 25 μm.

Figure 3. pSMAD1/5 regulates HFSC and TAC transcriptional networks

(A) Venn diagram of pSMAD1/5 ChIP-seq of HFSC and TAC chromatin, compared to RNA-seq profiling of transcripts ≥2X (p<0.05) changed in the two populations (“signature genes”). (B) SMAD1/5 occupies predominantly enhancer regions. (C) Overlapping pSMAD1/5 peaks for genes bound by pSMAD1/5 in both HFSC and TAC chromatin. (D) Unbiased pSMAD1/5 binding motif analysis. (E) Binding pattern of pSMAD1/5 on the Id1 locus and description of C/G(CAG)G/C mutations introduced. (F) Luciferase reporter assays show decreased BMP sensitivity of mutated Id1 C/G(CAG)G/C enhancer regions. (G) Gene Ontology (GO) analyses of pSMAD1/5 ChIP-seq targets (common or unique) for HFSCs and TACs. (H) Examples of HFSC signature genes bound by pSMAD1/5 and active in HFSCs, but silenced in TACs (I) Examples of TAC signature genes bound by pSMAD1/5 and active in TACs, but silenced in HFSCs. (J) Transcription Factor Binding Site analysis of pSMAD1/5 ChIP targets unique for either HFSCs or TACs reveal enriched binding motifs for putative lineage regulators.

Figure 4. Identification of BMP-sensitive pSMAD1/5-bound targets

(A) Venn diagram showing overlap between ORS, HG and TAC signatures and the mRNAs ≥2X changed in Bmpr1a cKO versus Ctrl HFSCs. (B) Venn diagram showing overlap between pSMAD1/5-bound genes and mRNAs ≥2X changed in HFSCs. (C) Relative mRNA expression of abundant BMP-sensitive pSMAD1/5 targets in cKO/WT. (D) Motif analysis of HFSC BMP-sensitive pSMAD1/5 targets based on previously published TF motifs (see Supplementary Methods) (E) Venn diagram showing the same analysis as in (B) but for TACs comparing to RNA profiling from IRS enriched or total TACs. (F) Unbiased GO analysis of 207 BMP-sensing pSMAD1/5 targets in TACs show enrichment of HF genes. (G) Differential expression of pSMAD1/5-bound, BMP sensitive targets in either IRS-TACs, HS-TACs or total TACs. (H) Enrichment of bHLH motifs was found in both HFSC and TAC targets, whereas only TAC BMP targets were enriched for GATA motifs.

Figure 5. ID proteins as novel mediators of HFSC quiescence

(A) pSMAD1/5 ChIP-seq targets ≥2X differentially expressed upon loss of Bmpr1A in HFSCs. Note that signature genes for quiescent HFSCs are downregulated while those for ORS cells are upregulated in Bmpr1a-null HFSCs. (B) ID1 and ID3 immunostaining in telogen HFs. (C) Verification of Id RNA profiling data. (D) Id1 ablation enhances bulge/HG proliferation at AnaI. (E) Overexpression (OE) of Id1 delays anagen progression. (F) Quantifications of EdU-incorporation in AnaII. (G) ID loss reduces and ID OE enhances proliferation in HFSCs 48 hrs after depilation to induce synchronized activation of hair cycling. (H–I) Id1 cKO mice subjected to repetitive depilation-induced hair cycles eventually fail to regrow hair coat (H) and lose HFSCs. (I). Data are represented as mean±SEM. * = p<0.05, ** = p<0.01, *** = p<0.001, Students t-test or Anova. Scale bar = 25μm

Figure 6. BMP suppress IRS-progenitors through IDs

(A and B) ID1 and ID3 immunostaining in TACs and differentiating IRS and cortical progeny. (C and D) Loss of IDs alters the relative contribution of TACs to their differentiated progeny within the HF. (E) Schematic illustration of the differentiated layers of mature HFs. (F) GATA3+ cells in HFs are diminished by Id1 OE and expanded upon Id1/Id3 ablation. (G and H) Quantifications of proliferating (EdU+) TACs in Id1/Id3 dKO HFs (J) or Id1 OE HFs. (I) Expression of bHLH proteins in HFSCs vs TACs show enrichment of HES/HEY proteins in TACs. (J) HES1⁺ TACs are reduced upon ID loss (Bmpr1a cKO) and expanded with ID OE. (K) Bmpr1a ablation in the total TAC pool leads to their failure to properly express differentiation-specific keratin genes of both IRS and cortical lineages (see also Table S7). Data are represented as mean±SEM. * = p<0.05, Students t-test or Anova. Scale bar = 25μm.

Figure 7. Model for how BMP signaling affects the HFSC lineage

HFSC quiescence is maintained in a BMP high/WNT low environment (Telo). Activation of stem cells in the HG requires combined BMP inhibition and WNT upregulation (AnaI). This leads to the emergence of the TAC pool which expresses SHH, and stimulates bulge HFSCs to self-renew and sustain ORS growth. SHH also stimulates DP to elevate BMP inhibitors and promote TAC expansion. In AnaIIIa, BMP signaling remains relatively low in the lower matrix, which commits to form GATA3⁺ Shh⁺IRS-TACs; we posit that SHH signaling in this pocket preferentially affects the adjacent (lower) DP, resulting in higher BMP signaling in the upper-matrix specifying ID⁺ HS-TACs. Terminally differentiating IRS upregulates BMP signaling; the differentiating cortex is in a complex milieu of DP, IRS and melanocytes, where they receive reduced BMP and elevated WNT signaling. In the differentiating medulla, there are no signs of WNT or BMP signaling, indicating that the two pathways are not always inversely coupled. However, the failure of this lineage to form in the absence of BMPR1A is consistent with a role for BMP signaling in forming their TACs.