Mammalian skin contains thousands of hair follicles, each undergoing continuous regenerative cycling. A hair follicle cycles through anagen (growth), catagen (involution) and telogen (resting) phases, and then re-enters anagen. At the base of this cycle is the ability of hair follicle stem cells to briefly exit their quiescent status to generate transient amplifying (TA) progeny, but maintain a cluster of stem cells. It is generally believed that a niche microenvironment is important in the control of stem cell homeostasis in various systems8
. Within a single hair follicle, periodic activation of β-catenin in bulge stem cells is responsible for their cyclic activity3
. However, how these stem cell activation events are coordinated among neighboring hairs remains unclear. It is possible that a population of hair follicles could cycle simultaneously, randomly, or in coordinated waves. We recently observed a “cyclic alopecia” phenotype in Msx2
null mice, which in essence represents coordinated hair regenerative activity in a population of follicles and is manifest as traversing hair waves9–11
(Supplementary Fig. S1
Classical works have documented hair growth waves in rats, mice, and other mammals12,13
. Opinions differ as to whether the hair growth pattern is controlled by local inherent rhythms, systemic factors or both. Since there is a period following anagen during which “the systemic stimulus is unable to exert an effect”, the concept of “telogen refractivity” was conceived14
. A substance, termed a “chalone”, which can inhibit anagen development, was proposed to explain this phenomenon15
. However, despite efforts to identify the “chalone”16,17
, its molecular nature has remained elusive for the past 50 years.
Intrigued by these dynamic, complex hair growth patterns (Supplementary Fig. S1
), we set out to find the underlying molecular mechanisms. A hair cycle domain is a region of skin which contains a population of hair follicles cycling in coordination. That such domains form implies the existence of signals that serve to spread and stop waves of hair growth. This prompted the suggestion that skin regions in telogen can be in either of the two functional phases: competent telogen which allows the anagen re-entry wave to propagate, and refractory telogen which arrests the wave (). We analyzed the cycling behavior of domains in more than 30 living mice (older than 2 months) for up to 1 year (Supplementary Fig. S1
), and consistently found that there is a minimal 28-days-long telogen phase (defined as the early telogen). Following this phase, telogen can either end right away (0 days) or persist for any number of days up to about 60 days. This phase (defined as the late telogen) contributes to the apparently highly variable telogen length ().
Defining refractory and competent telogen
This suggests the first 28 days of telogen are essential for the hair cycle and may represent the refractory phase. To test this idea, we used club hair plucking, which can induce hair regeneration. We gauged responses by the time required for regeneration to start after hairs are plucked (see Supplementary Methods
). When 50 hairs were plucked from early telogen skin, a longer time was required for hair growth than was the case when a comparable number of hairs were plucked during late telogen (requiring 42 vs 13 days). When 200 hairs were plucked, the time required for hairs to re-grow became shorter but still differed between early and late telogen (28 vs 9 days; , Supplementary Fig. S2
). Thus the functional status of a particular skin region can be determined by the hair plucking/regeneration assay. In the follicles we studied, early (up to 28 days) and late (after 28 days) telogen periods correlate well with refractory and competent telogen phases.
If the refractory and competent states of hair cycle domains are transient, then offsetting the timing of hair cycling in a localized region should lead to the formation of new hair cycle domains. We tested this by local application of cyclosporine A (a powerful anagen inducing agent which can overcome refractory telogen18
) to skin region about 10mm in diameter which was in telogen day 1. Eight days later, the treated region was in the induced new anagen, whilst the surrounding skin continued its progression through refractory telogen. Soon after the treated region completed anagen and re-entered early (new) telogen, the surrounding skin had progressed into late (competent) telogen. When a new hair growth wave approached, it propagated without obstruction over the untreated competent skin, but met resistance in the treated refractory region, thus forming a new hair cycle domain ().
Hair cycle domains are different from regionally specific domains specified differently in development (e.g., footpad vs dorsal paw). The exact domain boundaries can shift from cycle to cycle and the domain patterns become more complex as the mouse matures (Supplementary Fig. S1
). These complex hair cycle domains can be affected by systemic factors. For example, during pregnancy and lactation, female mouse hairs which enter telogen are unable to re-enter anagen. Thus multiple hair cycle domains are reset into one single domain after pregnancy/lactation19
(). Estrogen and prolactin have been implicated inhibition of anagen initiation (Supplementary Reference 1, 2
We then wanted to know the molecular mechanisms which constitute this refractoriness. Using in situ
hybridization and several lacZ reporter mice (including Bmp4-lacZ, Noggin-lacZ
, TOPGAL reporter), we searched for cyclic molecular expressions which correlate with refractory and competent telogen. In longitudinal sections of a hair cycle domain, the hair wave is “frozen” and successive temporal hair cycle stages are laid out in a spatial order11
, thus facilitating molecular analyses. We observed Wnt signaling and Msx2,
amongst others, to be expressed in different hair follicle compartments and to fluctuate with hair cycling as reported (Supplementary Fig. S6
). Unexpectedly, we observed the expression dynamics of inter-follicular Bmp2
to be out of phase with the dynamics of Wnt signaling (, Supplementary Fig. S5a–e
expression is absent in early anagen and gradually intensifies to reach its peak level in anagen V–VI. Bmp2
expression remains high in early telogen, but becomes absent in late telogen (, Supplementary Fig. S5c–e
). A long skin strip spanning two hair cycle domains shows two Bmp2
-expressing segments (). Bmp4
exhibits similar on and off expression dynamics as shown by semi-quantitative RT-PCR, in situ
hybridization () and Bmp4-lacZ
expression (Supplementary Fig. S3
). On the other hand, Noggin-lacZ
expression shows that on and off dynamics of mesenchymal noggin
(including dermal papilla and dermal sheath; Supplementary Fig. S4
coincides with the hair cycle rhythm. Since BMP activity can be modulated by multiple factors (different ligands, antagonists, and receptors), we measured BMP signaling output by immunostaining for p-SMAD 1/5/8. The results showed distinct periods of nuclear pSMAD activity, high in early/refractory and absent in late/competent telogen hair follicles ().
Periodic Bmp signaling in the dermis and subcutaneous adipose tissue
We noted that the ability to propagate anagen induction is limited to early anagen follicles. A wave front is halted when it faces a refractory telogen region. By the time this refractory telogen region progresses into competent telogen, the previously propagating anagen follicles have progressed into late anagen, and propagation does not resume (′, b″; Supplementary Fig. S5c, d
). Although the surrounding environment is now competent, late anagen follicles are unable to propagate. In this way, the traditional anagen period can be divided into early (anagen I–IV) propagating and late (anagen V, VI) autonomous anagen, with low and high Bmp2
expression respectively in these phases (, Supplementary Fig. S3, 5
). We summarize the rhythms of marker genes expression in . and Supplementary Fig. S7
Where are the Bmp producing cells? Interestingly, much of the periodically expressed Bmp2
transcripts are produced by subcutaneous adipocytes, as judged by double staining with Sudan Red (). Periodic expression of Bmp4
is seen in the intra-follicular epithelium, secondary hair germ, dermal papilla and adjacent extra-follicular dermal fibroblasts (Supplementary Fig. S3
). Collectively, we define the extra-follicular sources of periodic Bmp2/4
expression as the dermal macro-environment. On the other hand, Bmp-antagonizing noggin
appears to be constitutively expressed in bulge stem cells (Supplementary Fig. S4
). The macro-environmental Bmps may have major additive effect on strength of intrafollicular (micro-environmental) Bmp6 and Bmp4 signaling21,25
in regulating quiescence of pSMAD–positive bulge stem cells, although the specific mechanisms of this regulation remain to be investigated. Since the eventual anagen initiation requires activation of Wnt/β-catenin3,4
, there is competitive equilibrium between Bmp and Wnt signaling21
. Stem cells have to integrate the multiple signaling inputs from both the micro- and macro- environments in order to make the decision of activation.
It should be noted that the first telogen (around postnatal day 19) is very short and new anagen initiates quickly without detectable refractory telogen. Dermis acquires telogen refractivity with maturation and 2nd telogen (postnatal day 45–70; ′) does have it.
These findings lead us to hypothesize that: 1) in the “BMP ON” phase, the macro-environment prevents microenvironment-based activation of bulge stem cells (via Wnt signaling), resulting in refractory telogen; 2) in the “BMP OFF” phase, the macroenvironmental block is removed and the threshold for microenvironment-based activation of stem cells is low. This results in competent telogen; hair follicles are free to enter new anagen either by stochastic self-activation or upon facilitation by adjacent early anagen follicles. We tested both parts of this hypothesis by means of transgenic perturbation of Bmp signaling, skin transplantation, and administration of exogenous BMP4.
If Bmps play a causative role in conferring refractory status, we should be able to reduce the period of refractory telogen by down-regulating Bmp signaling. We did this by over-expressing noggin
under the control of a keratin 14 promoter in KRT14-NOG
. The minimal length of telogen was reduced down to 6 days, and the maximal length down to 11 days (). As a result, these mice display continuous propagation of hair regenerative waves and have highly simplified patterns of hair cycle domains (). We further tested the response of KRT14-NOG
hair follicles to hair plucking. The differences in response that we observed in WT mice in early vs. late telogen were eliminated in KRT14-NOG
mice. In all cases, plucked KRT14-NOG
hair follicles required only approximately 6 days to re-enter anagen (). Recently, the importance of Bmp activity in suppressing stem cell activity has also been shown by tissue specific deletion of BMP receptors21,23
Altered hair regenerative wave dynamics in KRT14-NOG mice and non-autonomous interactions with normal cycling host skin after transplantation
The currently-held concept of the stem cell microenvironment implies only autonomous regulation: thus the activation of stem cells depends only on signaling inputs from components intrinsic to the organ (here the hair follicle itself3
). To test directly whether the activation of stem cells is indeed subjected to non-autonomous regulation, we transplanted skin grafts from pigmented KRT14-NOG
mice onto albino SCID host mice. If the control of stem cells activation is intrinsic to the follicles, hair cycling behavior should remain the same for both donor and host. Instead, we observed donor-host interactions, reflecting a non-autonomous relationship, with the outcome dependent on the size of the transplanted skin graft. When a small graft of KRT14-NOG
skin (~1mm in diameter) was transplanted, the donor skin remained in telogen longer and could respond to an anagen activating wave originating from the host (). Thus we achieved functional rescue of KRT14-NOG
phenotypes. On the other hand, when a large skin graft (>10mm in diameter) was transplanted, the graft exhibited a greater degree of autonomous control. Within the graft, hair follicles continued to re-enter anagen rapidly. Providing evidence of a donor effect, host telogen hair follicles surrounding the perimeter of KRT14-NOG
skin graft acquired early competence, after only 11 days in telogen (vs. 28 days), and re-entered anagen (visible as a rim of white hairs) together with pigmented donor hairs ().
Classical experiments using skin graft transplantation to ask whether hair growth patterns are controlled intrinsically or systemically have produced different results14
. We have repeated autologous skin graft transplantation experiments and observed that hair growth patterns are initially intrinsic to the donor, but are gradually entrained to the host rhythm after several hair cycles (not shown). Consequently the discrepancy amongst classical experiments may be due to the size of the graft and the time chosen for readout. At the molecular level, our results demonstrate involvement of the Bmp pathway in the non-autonomous interactions among follicle populations. It remains to be investigated whether the process depends on the direct diffusion of Bmps and Bmp antagonists or is indirectly mediated by other mechanisms24
Finally, we tested whether a direct local delivery of BMP protein can convert competent telogen status to refractory in normal mice. hBMP4-soaked beads were implanted into competent telogen skin ahead of an anagen spreading wave (see Supplementary Methods17
). 12 days later, hBMP4, but not control BSA, prevented the propagation of the wave around the beads (). Thus the level of BMP activity can indeed explain the functional status (refractory vs competent) of a skin region.
The results presented here add new dimensions to our understanding of skin biology. Firstly, these findings demonstrate that in addition to short distance microenvironmental control17,25
, the activation of stem cells within large groups of neighboring hair follicles is subject to long distance macroenvironmental control from the surrounding dermis (). This concept is readily applicable to other organs. For example, while Bmp4
is constantly expressed in the mesenchyme of intestinal microvilli, bursts of noggin
expression in the villi stem cell niche may act to transiently lower BMP signaling, thus allowing stem cells to proliferate for epithelial renewal26
. Secondly, extrafollicular periodically expressed Bmp2/4 appear to fulfill the criteria of the elegant but elusive “chalone” proposed to explain patterned hair growth14,15,17
, thus solving a 50 year old puzzle. Thirdly, the dynamic expression of Bmp2
in dermal adipocytes suggests a link between two skin organ systems. Since subcutaneous fat, like hairs, has a thermo-regulatory function and leptin is present in the dermal papilla of hair follicles27
, periodically expressed Bmp2
may coordinate the function of these two organs in response to the external environment, and may have implications for the evolution of integuments28
. Fourth, the asynchronous cyclic expression of Bmps and β-catenin in the dermis and hair follicle provide a platform for mutual modulations of these “clocks” in the skin. They also imply that stem cell regeneration is subject to the control of biological rhythms.
Functional phases of the hair cycle
Finally, we note that mouse skin has been used extensively as a model in studies of carcinogenesis, intra-cutaneous drug delivery, and stem cell biology29,30
. Such studies are usually designed on the assumption that the skin is a stable and largely uniform medium. Our novel findings show clearly that this assumption is rarely if ever justified.