Regenerative hair waves in aging mice and extra-follicular modulators follistatin, dkk1, and sfrp4

Abstract

Hair cycling is modulated by factors both intrinsic and extrinsic to hair follicles. Cycling defects lead to conditions such as aging-associated alopecia. Recently, we demonstrated that mouse skin exhibits regenerative hair waves, reflecting a coordinated regenerative behavior in follicle populations. Here, we use this model to explore the regenerative behavior of aging mouse skin. Old mice (>18 months) tracked over several months show that with progressing age, hair waves slow down, wave propagation becomes restricted, and hair cycle domains fragment into smaller domains. Transplanting aged donor mouse skin to a young host can restore donor cycling within a 3 mm range of the interface, suggesting that changes are due to extracellular factors. Therefore, hair stem cells in aged skin can be reactivated. Molecular studies show that extra-follicular modulators Bmp2, Dkk1, and Sfrp4 increase in early anagen. Further, we identify follistatin as an extra-follicular modulator, which is highly expressed in late telogen and early anagen. Indeed, follistatin induces hair wave propagation and its level decreases in aging mice. We present an excitable medium model to simulate the cycling behavior in aging mice and illustrate how the interorgan macroenvironment can regulate the aging process by integrating both "activator" and "inhibitor" signals.

Figure 1. Aging mice show altered regenerative hair wave patterns: longer telogen and smaller domains

a. Twelve month old mouse was photographically documented for 358 days until it died. Mouse age (days) is indicated above each photograph. b. The telogen period (red dot) increases and the hair cycle domain (blue square) size decreases with advancing age. Error bar is shown. c. Average telogen period is longer in older compared to younger mice (n=5). d. Representative FACS scatterplots and summarized CD34+/CD49f + cell percentages are similar for young and old mouse epidermis. e. Immunostaining (green) of three exemplary hair follicle stem cell markers in 2 and 22 month old mouse dorsal skin sections. Few differences in cell number and expression levels are seen. DAPI staining (blue).

Figure 2. Young mouse skin macro-environment partially rescues hair cycling in aging skin

a. Young SCID mouse dorsum rescued hair growth and telogen retention of small, old skin transplants. Hair wave propagated throughout the graft and surrounding younger host skin in the next cycle (day 89 post-transplantation). b. Telogen retention was partially rescued for large transplants. During the secondary cycle (days 51, 61), hair wave propagated ∼3mm inward from the explant border; not reaching the graft center. Later (day 105), hairs formed toward the graft center, did not propagate to surrounding areas. c. Telogen remains long in the central, large, old skin graft but becomes shorter in the periphery (n = 3). d, e, Grafting older skin to younger mice decreased Bmp2 and increased follistatin. Size bar: 5mm.

Figure 3. Macro-environment extra-follicular modulators, Bmp2, Dkk1 and Sfrp4, are up-regulated in old mice

Whole mount in situ hybridization (WMISH) of dorsal skin stripes taken from 24 month old mice. Hair cycle stages are estimated based on propagating hair waves. a. Bmp2 and Wnt signaling pathway inhibitors, including Dkk1 and Sfrp4, should have been negative in the propagating anagen in the normal adult (e.g., 6 month old) mice. But they are expressed during this stage in the 24 month old mice. b. Quantitative RT-PCR from intra-dermal adipose tissues revealed of the skin from 24 months old mice show an overall up-regulation of inhibitors in all hair cycling phases, comparing to 6 month old young mice.

Figure 4. Extra-follicular follistatin promotes hair wave propagation in young mice, but levels decrease in older mice

a-e. Follistatin in young adult (6 month) mouse skin. a, Follistatin WMISH shows expression in competent telogen (C) and propagating anagen (P), but not in refractory telogen (R) nor autonomous anagen (A). Confirmation by (b) immunostaining and (c) RT-PCR. Red arrows, follistatin positive cells. d, e. Follistatin soaked beads induce precocious anagen re-entry (day 13) which propagates to surrounding HFs (day 17), to a maximum area (∼ 250 mm², day 22; n=3). Control BSA soaked beads show no effect. f. RT-PCR shows follistatin is decreased in C and P phases of old (24 months) mice. g. WMISH confirms decreased follistatin levels in 24 month old mouse P phase.

Figure 5. Decreased wavefront velocity in aging mouse skin revealed by the excitable medium model

a-d. Wavefront velocity decreases in an excitable medium description of the HF cycle. Plots of a. wavefront velocity, u, vs inhibitor production rate, J. b. wavefront velocity, u, vs activator production rate, α1. c. Cycling times vs inhibitor production rate, J. d. Cycling times vs activator activation rate α1. Time spent by an individual follicle in propagating anagen (P), autonomous anagen (A), refractory telogen (R) and competent telogen (C). e. Summary. Epidermal hair stem cell activation is regulated by intra-follicular and extra-follicular dermal activators / inhibitors. Dermal macro-environmental inhibitor levels increase in aging skin. f. Extra-follicular modulators in different hair cycle stages of young and old mouse skin. In old mice, decreased activators and increased inhibitors shorten the P phase and lengthen the R phase.