Analyses of regenerative wave patterns in adult hair follicle populations reveal macro-environmental regulation of stem cell activity

Abstract

The control of hair growth in the adult mammalian coat is a fascinating topic which has just begun to be explored with molecular genetic tools. Complex hair cycle domains and regenerative hair waves are present in normal adult (> 2 month) mice, but more apparent in mutants with cyclic alopecia phenotypes. Each hair cycle domain consists of initiation site(s), a propagating wave and boundaries. By analyzing the dynamics of hair growth, time required for regeneration after plucking, in situ hybridization and reporter activity, we showed that there is oscillation of intra-follicular Wnt signaling which is synchronous with hair cycling, and there is oscillation of dermal bone morphogenetic protein (BMP) signaling which is asynchronous with hair cycling. The interactions of these two rhythms lead to the recognition of refractory and competent phases in the telogen, and autonomous and propagating phases in the anagen. Boundaries form when propagating anagen waves reach follicles which are in refractory telogen. Experiments showed that Krt14-Nog mice have shortened refractory telogen and simplified wave dynamics. Krt14-Nog skin grafts exhibit non-autonomous interactions with surrounding host skin. Implantation of BMP coated beads into competent telogen skin prevents hair wave propagation around the bead. Thus, we have developed a new molecular understanding of the classic early concepts of inhibitory "chalone", suggesting that stem cells within the hair follicle micro-environment, or other organs, are subject to a higher level of macro-environmental regulation. Such a novel understanding has important implications in the field of regenerative medicine. The unexpected links with Bmp2 expression in subcutaneous adipocytes has implications for systems biology and Evo-Devo.

Fig. 1. Growth of individual hairs vs. growth of hairs in populations

(A) Within one mouse cage each adult Msx2 null mouse displays a different pattern of hair growth. Hair growth patterns can be easily monitored in Msx2 null mice due to the cyclic alopecia phenotype. (B,C) Schematic illustration of hair cycling in one single hair follicle (B) versus cycling of a population of hair follicles (C). (D,E) In mice pelage hair follicles can sometime cycle individually (D), yet most of the time they cycle as coordinated waves (E). Anagen hair follicles are black. The position of telogen hair follicles is marked with sebaceous glands stained in red with Oil Red.

Fig. 2. Long term follow up of changing hair cycle domains on dorsal skin of the same mouse

Changes in the coat of a single Msx2 null mouse with cyclic alopecia were followed over 145 days. Pictures were taken every 2-3 days and selective ones are shown here. Next to each picture there is a schematic drawing of the hair growth pattern. Key patterning events are annotated. From Plikus et al., 2008.

Fig. 3. Regenerative hair wave and hair cycle domains

(A) Schematically, a hair cycle domain is composed of an initiation center, a spreading wave, and a boundary. Blank arrows, direction of the spreading waves. An, anagen; T, telogen. Roman numerals show anagen stages in accordance to Muller-Rover et al., 2001. (B) On the mouse back, the hair cycle domain is spreading both cephalically and caudally. T, telogen. (C,D) View of inverted skin clearly shows hair cycle stages. Areas encircled in C are enlarged in (D). (E,F) Longitudinal sections of the skin corresponding to the whole mount skin strip preparation in panel 3C is shown. Note the difference in the thickness of the skin stripe at the different hair cycle stages. Also see Suzuki et al., 2003. (G) In C3H/HeJ agouti mice, the hair fiber pigmentation is yellowish in the distal (eumelanin pigmentation) but black in the more proximal region (pheomelanin pigmentation). This helps us visualize the molting line (flanked by red dots), or the wave front of the hair cycle domain. Modified from Plikus and Chuong, 2008. Size bars: (C) 5 mm; (D) 500 μm; (E) 1 mm; (F) 200 μm.

Fig. 4. Spatial and temporal changes of Bmp2 expression in the regenerating hair wave

In situ hybridization of Bmp2. From Plikus et al. (2008). (A-E) Dynamic expression of Bmp2 in a spreading hair cycle domain. (A) Different hair cycle stages are spread spatially on a longitudinal skin strip. In situ hybridization for Bmp2 (green). Blank arrows, the direction of the spreading waves; —| sign, boundary between anagen (An, which was competent telogen) and refractory telogen (Tel). Two boxed regions showing wave front and boundary are enlarged in panels (B,C). Also note the change of skin thickness. (B,C) Bmp2 is negative in the wave front region, including competent telogen and propagating anagen. Extrafollicular Bmp2 starts to appear around anagen IV and its expression becomes stronger in anagen VI (yellow arrows). Extrafollicular Bmp2 persists into the refractory telogen stage (red arrows). (D) Progressive changes of (C). Entering late telogen, the region to the right of the boundary becomes Bmp2-negative (green arrows) and competent to enter anagen. However, anagen VI (yellow arrow) follicles do not send out spreading signals and the boundary remains stable. (E) A telogen skin strip shows Bmp2 expression during early and refractory phases (red arrows), but lack thereof during late and competent phase (green arrows). Scale bars, (A) 1 mm; (B-E) 500 μm. From Plikus et al., 2008.

Fig. 5. The temporal oscillation and spatial distribution of Bmp signaling pathway members throughout the hair cycle is summarized

(A) Temporal expression of Bmp4, Bmp2, noggin and a putative Bmp activity reporter (see Fig. 8). Solid area, strong expression; striped areas, expression is lost from some but not all sites. (B) Schematic summary of multiple expression sites of noggin, Bmp4 and Bmp2 during functional phases of the hair cycle. From Plikus et al., 2008.

Fig. 6. Correlation of refractory telogen and Bmp2 expression

A long skin strip spanning two hair cycle domains shows two Bmp2-expressing segments which also correspond to the refractory telogen region demonstrated by observation.

Fig. 7. Spatial and temporal changes of putative Bmp reporter activity in the regenerating hair wave

(A) 52bp Msx2 promoter region contains both Smad and homeodomain consensus sites. (B-G) Summed putative Bmp activity may be estimated in vivo using 52bpMsx2-hsplacZ transgene expression. However, this 52 bp also contain a homeobox binding site which can affect Bmp reporter activity. (F,G) Positive trans-gene expression is seen starting from late anagen. Early anagen hair follicles (red arrowheads on F; yellow arrowheads on G) do not show 52bpMsx2-hsplacZ transgene expression. Expression of 52bpMsx2-hsplacZ transgene during telogen is dynamic: it is expressed during early telogen (refractory phase; B,D), but absent during late telogen (competent phase; C,E). (H) Putative Bmp reporter activity is high in refractory but low in competent telogen follicles. (I) Left panel: Putative Bmp reporter activity (blue color) and keratin 15 immuno-localization (brown) positive cells partially overlap (red arrow). Right panel: Similarly, some Bmp reporter activity (blue color) and pSMAD positive cells (brown) overlap (yellow arrow), yet others are positive for either Bmp activity or pSMAD (white arrow) only.

Fig. 8. Diversity of hair cycle domain patterns in transgenic mice

(A) Msx2 null mice form more complex and dynamic patterns than control mice. (B) Krt14-Nog mice form simplified patterns without lateral and central domains. They also do not display stable domain borders, but rather show continuous advancement of regenerating transverse waves. From Plikus et al., 2008.

Fig. 9. (Left). Illustration of transplantation experiment

Transplanting Krt14-Nog skin to SCID mice allowed us to study the interaction between skins with either normal or high levels of noggin. (A) When small transplants were used, the refractory telogen phase in Krt14-Nog skin can be partially restored by the surrounding host skin. Red stop signals represent refractory signals that can affect the donor skin. (B) When bigger transplants were used, Krt14-Nog skin can initiate propagation waves spreading in the host skin which is in a competent telogen phase, or to ≪neutralize≫ the refractory telogen region of the host up to 2.8 mm deep. Green arrows represent the spreading signals. The experiments suggest the interactions are non-autonomous (Plikus et al., 2008). Whether this effect is mediated through direct diffusion or an indirect relay remains to be determined.

Fig. 10. (Right). Altered activity of putative Bmp reporter in Krt14-Nog mice

(A) Premature loss of putative Bmp activity during early telogen in Krt14-Nog / 52bp Msx2-hsplacZ mice. (B) Restored 52bp Msx2-hsplacZ transgene activity in small Krt14-Nog skin flaps transplanted on SCID mice. In the Krt14-Nog / 52bp Msx2-hsplacZ skin, there is no Bmp activity. (C) Upon transplantation however, putative Bmp activity is restored in the telogen follicles. From Plikus et al., 2008.

Fig. 11. BMP protein can convert competent telogen status to refractory

(A) Human BMP4-soaked beads caused hair propagation wave (green arrowed curve) to go around them. (B) Albumin does not have this effect. Red broken line, domain border. From Plikus et al., 2008.

Fig. 12. Functional phases of the hair cycle

Schematic representation of classical hair cycle stages (inner black and white circle; modified from Stenn and Paus, 2001) and the new functional phases revealed in our studies (colored outer circle). Based on the ability of anagen follicles (gray) to send out signals to induce adjacent hair follicles (by reduction of Bmp activity) in anagen I - IV, not anagen V and later (black), we propose to name these sub-phases propagating anagen (blue) and autonomous anagen (yellow). Based on the ability of telogen follicles (white) to respond or not respond to regenerative signals, we name these sub-phases refractory telogen (red) and competent telogen (green). Modified from Plikus et al., 2008.

Fig. 13. Schematic illustration of the relationship between macroenvironment and micro-environment of hair follicles

The inter-follicular dermal macroenvironment includes 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 codes are similar to Fig. 12. From Plikus et al., 2008.