Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration

Abstract

In the age of stem cell engineering it is critical to understand how stem cell activity is regulated during regeneration. Hairs are mini-organs that undergo cyclic regeneration throughout adult life, and are an important model for organ regeneration. Hair stem cells located in the follicle bulge are regulated by the surrounding microenvironment, or niche. The activation of such stem cells is cyclic, involving periodic beta-catenin activity. In the adult mouse, regeneration occurs in waves in a follicle population, implying coordination among adjacent follicles and the extrafollicular environment. Here we show that unexpected periodic expression of bone morphogenetic protein 2 (Bmp2) and Bmp4 in the dermis regulates this process. This BMP cycle is out of phase with the WNT/beta-catenin cycle, thus dividing the conventional telogen into new functional phases: one refractory and the other competent for hair regeneration, characterized by high and low BMP signalling, respectively. Overexpression of noggin, a BMP antagonist, in mouse skin resulted in a markedly shortened refractory phase and faster propagation of the regenerative wave. Transplantation of skin from this mutant onto a wild-type host showed that follicles in donor and host can affect their cycling behaviours mutually, with the outcome depending on the equilibrium of BMP activity in the dermis. Administration of BMP4 protein caused the competent region to become refractory. These results show that BMPs may be the long-sought 'chalone' inhibitors of hair growth postulated by classical experiments. Taken together, results presented in this study provide an example of hierarchical regulation of local organ stem cell homeostasis by the inter-organ macroenvironment. The expression of Bmp2 in subcutaneous adipocytes indicates physiological integration between these two thermo-regulatory organs. Our findings have practical importance for studies using mouse skin as a model for carcinogenesis, intra-cutaneous drug delivery and stem cell engineering studies, because they highlight the acute need to differentiate supportive versus inhibitory regions in the host skin.

Figure 1. Defining refractory and competent telogen

(a) Propagation (blank arrow) of hair regenerative waves is seen in Msx2 null mice (also see supplementary Fig. S1). Similar patterns can be seen in normal black mice after hair clipping. Roman characters, anagen stages; T, telogen. (b) Under physiological conditions, some domains can become refractory to the spreading wave. (c) The durations of anagen and telogen were measured in 22 hair cycle domains from dorsal and ventral skin. (d) Experimental induction of refractory telogen with cyclosporine A (cyclA). X coordinate represents time scale (in days) when experiments began in the early telogen of the non-treated skin region. CyclA was applied to a localized region (treated, Tx) during early telogen and induced new anagen at about 8 days later. The surrounding non-treated refractory telogen skin (Non Tx) remained in telogen. When the non-treated skin was at day 19 of their telogen, treated Tx skin already proceeded to the late stage of its induced new anagen (panel d′, day 19). When non-treated skin was at day 24 of their telogen, cyclosporine treated region had finished its induced new anagen phase and entered new telogen (panel d″, day 24). Soon the non-treated skin progressed into the competent telogen. At day 34, non-Tx region entered its natural anagen. The regenerative wave spread but can not enter Tx region because it is still in its refractory telogen period (panel d‴, day 37). Black, anagen; green, competent telogen; red, refractory telogen. (e) In female mice, multiple hair cycle domains were reset into one after pregnancy/lactation. (f) Hair plucking/regeneration was used to gauge competent and refractory telogen status (n=16). The minimum time (shown in days) represents the time required for new pigmented hair filaments to be visible. This time is shorter when more hairs were plucked or when the same number of hairs were plucked in competent period.

Figure 2. Periodic Bmp signaling in the dermis and subcutaneous adipose tissue

(a) Different temporal stages are spatially laid across the skin strip. The dark field illumination shows hair follicles (white) and Bmp2 in situ hybridization (green). Note the two are out of phase. An, anagen; Comp, competent; Refr, refractory; Tel, telogen. Open arrows, the direction of the spreading waves; --| sign, boundary between anagen and refractory telogen. (b, b′) Enlarged regions from boxed region of a. It shows details of Bmp2 expression during anagen spreading (b) and on the border of anagen VI – refractory telogen (b′). (b″), While later refractory telogen region becomes competent, anagen VI follicles still do not propagate. (c) A long skin strip includes two segments of dermal Bmp on and off expression. (d, e) Bmp2 and Bmp4 expressions are detected by semi-quantitative RT-PCR and in situ. (f) pSMAD immuno-staining is present in follicular epithelium, including bulge area (insert) and adjoining infundibulum (green arrow). (f′) Since skin in its second telogen phase (45–70 day after birth) is usually used in hair follicle and carcinogenesis studies, we show results at this stage, which are consistent with older mice. (g) Bmp2 expression (blue) co-localized within some Sudan red-positive adipocytes (red). (h) Schematic summary of the hair cycle rhythm (black) and newly identified dermal rhythm (red). Together, they define 4 new functional stages. Catagen is omitted for simplification. Scale bars: a: 1 mm; b: 500 um; e–g: 200 um.

Figure 3. Altered hair regenerative wave dynamics in KRT14-NOG mice and non-autonomous interactions with normal cycling host skin after transplantation

(a) Control and (a′) KRT14-NOG mice. Hair cycle domains in two different stages are shown, together with schematic domain boundaries generated by similar analysis used in supplementary Fig. S1. (b) Measurements show both refractory and competent telogen are shortened in KRT14-NOG mice (green bars). (c) Plucking/regenerative response in KRT14-NOG (green bars) is about 5 times faster. (e) When a small KRT14-NOG skin graft was transplanted into SCID skin, hair growth (e) and duration of refractory telogen (d) were partially rescued. (f) When a large KRT14-NOG skin graft (>10mm) was transplanted, it caused reduction of refractory telogen by inducing a rim of white hair from the host. (g, h) hBMP4-soaked beads caused hair propagation wave (green arrowed curve) to go around them, creating a new telogen domain. Albumin does not have this effect. Red broken line, domain border. Scale bars: e, g, h: 1 mm.

Figure 4. Functional phases of the hair cycle

(a) Illustration of the bulge niche microenvironment and inter-follicular dermal macroenvironment, including dermis, subcutaneous fat and adjacent follicles. Anagen stimulating (black and green) or inhibitory (red) activities are depicted with colored arrows. Follicles are in different stages: A, refractory telogen; B, competent telogen; C, propagating anagen; D, autonomous anagen follicles. Blue circle in A, intra-follicular micro-environment. Color coded similar to panel b. (b) New functional phases (colored outer circle) mapped against classical hair cycle stages (black and white inner circle). Based on the growth-inducing ability of follicles, anagen is divided into propagating (inducing, blue) and autonomous (non-inducing, yellow) phases. Based on ability to respond to regenerative signals, telogen is divided into refractory telogen (red) and competent (green) phases.