Hair follicle stem cells and melanocyte stem cells remain quiescent within their hair follicle niche for weeks, a period known as telogen phase. With each new hair cycle, these two stem cell populations are coordinately activated. This happens when inhibitory signals are counteracted by activating cues that accumulate from Wnt and BMP/TGFβ (bone morphogenetic protein/transforming growth factor β) crosstalk with dermal papilla at the niche base6–8
. Synchronized activity continues throughout the hair cycle. During the growth phase (anagen), melanocytes at the base of the mature hair follicle (‘hair bulb’) produce and transfer pigment to neighbouring committed hair follicle stem cell progeny (‘matrix’) as they differentiate into hair cells2,5
.When destruction (catagen) ensues, melanocytes and matrix cells in the hair bulb apoptose, and the dermal papilla (enveloped by the hair bulb during anagen) retracts upward, returning the follicle to telogen. As anagen begins and a new hair bulb emerges, both hair follicle stem cells and melanocyte stem cells contain nuclear β-catenin, implicating canonical Wnt signalling in stem cell coordination6,8
. These and several other insights7,9,10
suggest how local environmental signals synchronize proliferation and lineage progression of stem cells during hair cycling.
Uncoupling melanocyte and epithelial stem cell behaviours occurs under transient conditions, that is, in response to ultraviolet radiation, and in various disease and injury states11,12
. Given the impact of Wnt and other signals on stem cells and their lineages, and current dogma that matrix cells must differentiate for melanocyte pigment to transfer10
, the mechanisms by which melanocyte stem cells can be selectively mobilized from their niche without otherwise disrupting the normal hair cycle remains unknown.
Our venture into this study was prompted by our finding that relative to progeny, hair follicle stem cells express elevated nuclear factor I/B (NFIB)1
. NFIB is required for lung and brain development and is often amplified and/or found at oncogenic chromosomal breakpoints in epithelial cancers13–15
. NFIB was first detected in epidermis at embryonic day 14.5 (E14.5), concomitant with upregulation of established skin progenitors. Expression intensified as hair follicle stem cells emerged ( and Supplementary Fig. 1a–c
Conditional Nfib targeting in hair follicle stem cells does not perturb hair cycle or follicle architecture
In adult hair follicles, NFIB co-localized with hair follicle stem cells, whose niche in telogen is subdivided into an upper ‘bulge’ compartment and lower ‘hair germ’ (or secondary hair germ) adjacent to dermal papilla (Supplementary Fig. 1d
). During anagen, NFIB-positive cells were also found within the upper outer root sheath (ORS), which in early catagen will form the new niche (bulge and hair germ) for the next hair cycle16
(). NFIB waned in transit-amplifying (TA) matrix progenitors ( and Supplementary Fig. 1e
). NFIB was not detected in melanocyte stem cells marked by dopachrome tautomerase (DCT) and tyrosine kinase receptor KIT in the upper ORS and bulge/hair germ, nor in differentiated melanocytes within the hair bulb17
(). Overall, both inside and outside the niche, hair follicle stem cells and melanocyte stem cells showed synchronized behaviours but distinct expression patterns.
To address the function of NFIB, we conditionally induced Nfib
ablation (cKO) in mouse hair follicle stem cells by using Sox9-CreER
. Unless specified, data are fromSox9-CreER
mice, but both gave similar results. NFIB immunofluorescence verified that Nfib
was efficiently targeted in hair follicle stem cells, consistent with K15
expression primarily in ORS/bulge/hair germ; furthermore, when Rosa26floxSTOPfloxYFP
; yellow fluorescent protein (YFP)) was used to mark and trace the progeny of Cre-induced cells, only hair follicle stem cells and their subsequent progeny, and not KIT+
melanocytes or dermal cells, were fluorescently labelled18
( and Supplementary Fig. 2a–c
). Despite efficient targeting, however, Nfib
-cKO hair coats and hair cycling appeared normal ().
Equally surprising to the absence of hair follicle stem cell lineage defects were melanocyte lineage abnormalities. Fontana–Masson staining revealed atypical melanogenic cells at the niche base of telogen hair follicles ( and Supplementary Fig. 2d
). Immunostaining showed increases in melanocytes (KIT+
) throughout hair germ and bulge, and ectopic presence of differentiated melanocytes (KIT+
) within hair germ ( and ).
NFIB loss enhances melanocyte stem-cell self-renewal and perturbs melanocyte stem cells activity in the hair follicle stem cell niche
A priori, ectopic differentiated melanocytes in cKO hair germs might reflect hair bulb melanocytes that somehow escaped apoptosis during catagen. Alternatively, they could arise from precocious differentiation of niche melanocyte stem cells. To distinguish between these possibilities, we analysed melanocyte behaviour at each hair cycle stage ( and Supplementary Fig. 4
). During anagen, TYRP1+
cells dropped to wild-type levels in the bulge/upper ORS. As dermal papilla returned to the niche during late catagen, TYRP1+
numbers again soared, persisting until the next anagen. By contrast, control stem cell niches displayed much more modest melanocyte stem cell differentiation, which occurred at telogen→anagen rather than catagenRtelogen. The difference in this timing was >3 weeks for young adult mice.
Accompanying the disappearance of differentiated melanocytes within the anagen Nfib
-cKO bulge/upper ORS, was their appearance in the hair bulb (upper matrix) ( and Supplementary Fig. 4
). Within the bulb, pigment transfer to differentiating hair cell recipients seemed normal. Additionally, at the start of catagen, TYRP1+
melanocytes underwent apoptosis and were eliminated by mid-catagen in both control and cKO hair bulbs. In contrast to differentiated melanocytes, TYRP1−
melanocyte stem cells remained elevated throughout cKO hair cycles, where they resided in the bulge/upper ORS. The only time at which melanocyte stem cells dropped transiently was during late catagen/telogen, when melanocyte stem cells near dermal papilla precociously differentiated.
These results pinpointed defects to the stem cell niche; furthermore, dermal papilla proximity seemed to affect primarily the uncoupling and premature differentiation of melanocyte stem cells rather than self-renewal (). In addition, melanocyte stem cells in anagen stem cell niches were still negative for TYRP1 even in 1-year-old Nfib-cKO hair follicles (), indicating that ectopic differentiated melanocytes from telogen do not accumulate within the niche over hair cycles.
To test melanocyte stem cell activity further, we mated Nfib
-cKO to Dct-EGFP
mice and isolated EGFP+
bulge cells from telogen hair follicles. Melanocyte differentiation markers, for example, Kit
, were upregulated in EGFP+
cells, whereas genes expressed by both melanocyte stem cells and differentiated melanocytes showed no change (Supplementary Fig. 3d
). Finally, hair coats of ageing cKO mice were still pigmented, and melanocyte stem cell levels were sustained ( and Supplementary Fig. 3e
). These data provided compelling evidence that NFIB-deficiency in hair follicle stem cells affects the timing of melanocyte differentiation without compromising melanocyte stem cell biology and/or function, which results in hair greying6,7,9,11
Ultrastructural analysis unveiled new defects within the stem cell niche ( and Supplementary Fig. 5
). Niche melanocytes closest to dermal papilla had pigment granules and immature melanosomes. Surprisingly, however, adjacent hair germ cells also had pigment granules. Their epithelial identity was shown by keratin filaments, desmosomes and hemidesmosomes. Although poorly understood, hair cell uptake of pigment from differentiated melanocytes is thought to depend on FOXN110
, expressed by differentiating matrix cells but not by hair follicle stem cells19
Premature transfer of pigment promotes apoptotic cell death in hair follicle stem cells in the NFIB-deficient niche
Inappropriate accumulation of pigment proved calamitous for quiescent hair follicle stem cells in the telogen hair germ: it elicited their apoptotic death, typified by condensed chromatin, mitochondrial destruction, and cleaved caspase-3-immunolabelling ( and Supplementary Fig. 6
). In addition, unaffected neighbouring K5-positive hair germ cells proliferated ( and Supplementary Fig. 6
). Although hair germ proliferation is a normal sign of telogen→anagen8
, precocious hair cycle entry was not observed.
Apoptotic and proliferative defects within the niche disappeared in anagen (), concomitant with movement of differentiated melanocytes from niche to hair bulb (). These results indicate that apoptosis and hyperproliferation of Nfib-null hair follicle stem cells depend upon precocious differentiation of neighbouring melanocyte stem cells. They also indicate that when differentiated melanocytes inappropriately bequeath pigment to hair follicle stem cells rather than their customary differentiated progeny (hair cells), pigment-laden hair follicle stem cells are unable to cope, whereas healthy hair follicle stem cell neighbours proliferate to restock the niche.
Notably, in older Nfib
-cKO mice, black-pigmented dermal cells swarmed hair follicles and were even visible from the skin surface (Supplementary Fig. 7
). Although dermal melanoblasts exist in certain skin regions such as the ear, and can differentiate under some conditions20
melanocytes were found in cKO dermis. Rather, a number of cells encompassing this pigment were Mac1+
with features of macrophages. Irrespective of whether pigment-laden vacuoles within dermal cells reflected engulfment of dying, pigmented cells, or direct pigment transfer from melanocytes, these defects made the normalcy of the hair coat of ageing cKO mice all the more remarkable.
To dissect the molecular miscommunication between hair follicle stem cells and melanocyte stem cells in the niche, we used high throughput RNA-seq to transcriptionally profile bulge and hair germ hair follicle stem cells. Fluorescence activated cell sorting (FACS) of skins from K15-CrePGR/RosaYFP/Nfibfl/fl
(cKO) and Nfibfl/+
(het) mice were used for purifying CD34+
-hair germ cells (both YFP+
) (Supplementary Figs 2a and 8a
Of 800–1,000 messenger RNAs changed by≥twofold in NFIB-deficient hair follicle stem cells relative to control, 145 were upregulated and 99 were downregulated (Supplementary Figs 8b and 9
and Supplementary Table 1
). Quantitative PCR (qPCR) of independently purified samples validated the differences (Supplementary Fig. 8c, d
). Notably absent from the list were Foxn1
and derivatives of Pomc
—all previously implicated in melanocyte differentiation and/or pigment transfer10,12,21
. Also absent were genes involved in BMP/TGF-β signalling, known to function in stem cell niche quiescence. Similarly, NFIB loss did not seem to affect canonical Wnt signalling, a key stimulus for stem cell activity and fate commitment: Wnt-sensitive target gene Axin2
was unchanged in NFIB-deficient hair follicle stem cells, and upregulated genes included both negative and positive Wnt regulators.
These results were consistent with the normal hair cycle displayed by Nfib
-cKO skin (). Had any of these signalling pathways been perturbed, both stem cell populations—not just melanocyte stem cells—should have been affected. Moreover, hair follicle stem cells from mice genetically defective for Wnt, BMP and TGF-β signalling still expressed NFIB protein and mRNA (Supplementary Fig. 10a, b
). Thus at least within the quiescent stem cell niche, these signalling pathways seemed to be refractory to loss of NFIB, and NFIB seemed to be refractory to these signalling pathways.
To identify direct NFIB target genes, we performed chromatin-immunoprecipitation with high-throughput-sequencing (ChIP-seq) analysis on 10–15 million bulge hair follicle stem cells FACS-purified from 15–20 mice19
. Applying immunoprecipitation-grade NFIB antibody to chromatin, we identified 1,449 genes that were directly and reproducibly bound by NFIB ( and Supplementary Table 2
). NFIB-bound genes included Krt5
(Supplementary Fig. 11a
). Intriguingly, like Krt5
and its transcription factor-AP2 family regulators, NFIB was absent in most areas of Trp63
-null skin (Supplementary Fig. 10c
). Additionally, Nfib
is bound by p63 (ref. 22) and harbours TFAP2 binding motifs (data not shown), suggesting possible connections to these early stem cell markers.
RNA-seq and ChIP-seq analyses identify Edn2 as a direct NFIB regulated gene mediating inter-stem cell crosstalk
NFIB peaks were enriched in±2 kilobases (kb; 12%) and 2–50 kb (29%) sequences proximal to gene transcription initiation sites (Supplementary Fig. 11b, c
). A de novo
motif search identified five sequences within these peaks (Supplementary Fig. 11d, e
). Most common were TGGCA/T
GCCA, which when combined, comprised a palindromic motif. Notably, NFIB protein self-dimerizes, and its preferred binding motif is a TTGGCANNNTGCCAA palindrome23
. Moreover, in response to NFIB loss, 201 (~14%) NFIB target genes were differentially expressed relative to control hair follicle stem cells (Supplementary Fig. 12
). Of these, 44% were upregulated whereas 56% were downregulated in NFIB-deficient hair follicle stem cells. NFIB’s lack of apparent bias for hair follicle stem cell gene activation differed from the role of NFIC in cultured fibroblasts23
Searching for candidates whose altered expression might enhance melanocyte stem cell proliferation and differentiation, we focused on the 33NFIB-binding genes≥twofold up- or downregulated in bulge and hair germ hair follicle stem cells when Nfib is ablated (). Notable was the gene Edn2, encoding endothelin-2. Within the Edn2 promoter was an NFI-binding palindrome sequence containing an optimal spacer (). In both ChIP-seq replicates, NFIB bound to this site.
Endothelins are secreted factors with the ability to mediate intercellular crosstalk. All three endothelins stimulate cultured melanocyte stem cells5
. Although Edn3
is required for neural crest migration and melanocyte specification during embryogenesis24
, and Edn1
has been implicated in Wnt-mediated melanocyte proliferation6
, neither Edn3
appeared on our list of hair follicle stem cell genes bound by NFIB. Moreover, as assessed by RNA-seq and qPCR, Edn3
was not expressed in hair follicle stem cells, and Edn1
showed low expression and little change upon NFIB loss ( and Supplementary Table 1
). By contrast, at telogen→anagen, Edn2
was transiently activated inwild-type hair follicle stem cells19
, and in Nfib
-null hair follicle stem cells, Edn2
was upregulated more than twofold relative to controls. Pan anti-endothelin immunolabelling was also stronger in Nfib
-null relative to control hair follicle stem cells (). Finally, hair follicle stem cells express endothelial converting enzyme (ECE1) necessary to process/activate EDN2.
changes were cell-autonomous, because Edn2
mRNA was also upregulated in cultured Nfib
-null keratinocytes (Supplementary Fig. 13
). Moreover, this difference depended upon NFIB, as it was rescued by expressing the main keratinocyte isoform, NFIB3. By contrast, even though KIT ligand (Kitl
)mRNA seemed to be modestly induced in Nfib
-null hair follicle stem cells (Supplementary Table 1
), the levels were not influenced by NFIB3-rescue (Supplementary Fig. 13
), nor did we observe NFIB binding to the Kitl
promoter/enhancer in ChIP-seq analyses. Interestingly, however, whereas KITL was not detected in hair follicle stem cells, it was seen in dermal papilla17
Together, these results indicated a model whereby Edn2 induction by Nfib-null hair follicle stem cells enhances proliferation of neighbouring melanocyte stem cells and sensitizes them to precociously differentiate when they encounter KITL and possibly additional signals from dermal papilla. If true, then blocking KIT signalling should ameliorate precocious melanocyte stem cell differentiation in cKO hair follicles, whereas elevating EDN2 in wild-type hair follicle stem cells should generate phenotypic features of NFIB-deficiency.
We first tested this hypothesis by injecting a KIT-receptor-blocking antibody into the skins of cKO mice beginning in late catagen. By telogen, marked reductions were seen in TYRP1+
melanocytes and in activated-CASP3+
hair follicle stem cells (). Importantly and in agreement with previous findings17,25
, KIT inhibition in anagen hair follicles only affected lineage-committed proliferation and differentiation: undifferentiated melanocyte stem cells remained elevated in the cKO niche ( and Supplementary Fig. 14
We next induced EDN2 in wild-type adult skin by co-transducing E9.5 mouse embryos in utero
with high-titre lentiviruses26
harbouring: (1) a tetracycline-regulatable transactivator (rtTA
) coupled in a bicistronic transcript to EGFP
; and (2) H2BmRFP1
and a tetracycline-inducible promoter driving either Edn2
) or nothing (TRE
-only) (). Following selective EDN2 induction (Supplementary Fig. 15
-transduced hair follicle stem cell niches contained increased KIT+
cells and TYRP1+
differentiated melanocytes (). Notably, de novo
melanogenesis was not detected in similarly transduced epidermis, indicating that EDN2’s effects were confined to locations where preexisting melanocyte stem cells reside (data not shown). Finally, premature melanocyte stem cell differentiation in Nfib
-cKO niches depended upon EDN2, because marked reductions in TYRP1+
melanocytes and activated-CASP3+
hair follicle stem cells were observed when an endothelin receptor inhibitor (BQ788) was injected intradermally into cKO skin from late-catagen ().
In summary, our findings expose an unexpected gatekeeper, NFIB, which governs activity within the quiescent stem cell niche of hair follicles. Upregulation of a singleNFIB target, Edn2, seemed to be sufficient to uncouple coordinated stem cell behaviour. In contrast to all known genetic pathways perturbing melanocyte stem cell function within hair follicles, precocious melanocyte differentiation and ensuing chaos was cyclical and did not compromise stem cell pools, hair pigmentation or growth, even in ageing animals. The mechanism underlying this unprecedented phenotype lends new importance to the two-tiered structure of the hair follicle stem cell niche, and to dermal papilla/KITL-independent and dependent steps in controlling the effects of EDN2.
Endothelin 2 has not been implicated hitherto in normal cutaneous melanocyte physiology. Moreover, whereas melanocyte specification during embryogenesis fails to occur without Edn3
(ref. 27), melano-genesis is seemingly normal in Edn1
. This has left it unclear as to whether endothelins function postnatally. Similarly to Edn3
, elevating Edn2
in epidermis did not promote melanogenesis20
; however, our results show that when provided with the proper microenvironment and additional stimulatory signals, endothelins can influence adult melanocyte stem cells and their differentiation.
In closing, our findings add new understanding to how melanocyte stem cell and hair follicle stem cell behaviours maintain reliance upon cooperative factors within the niche. They also reveal how this communication might be selectively uncoupled in injury and disease states. Notably, Edn2
is induced upon ultraviolet irradiation and other stress conditions associated with increased pigmentation29,30
. Although beyond the scope of the present study, testing the possible role of NFIB in skin cancers, wound repair and stress responses merit investigation, as does removing possible redundancy from other NFI members expressed by hair follicle stem cells. In the future, it will be interesting to see the extent to which Nfib
downregulation will tip the balance from coupled to uncoupled states in health and disease. Our studies here emphasize the importance of endothelins as important messengers to uncouple melanocyte and hair follicle stem cell synchrony.