Direct migration of follicular melanocyte stem cells to the epidermis after wounding or UVB irradiation is dependent on Mc1r signaling

Abstract

During wound healing, stem cells provide functional mature cells to meet acute demands for tissue regeneration. Simultaneously, the tissue must maintain a pool of stem cells to sustain its future regeneration capability. However, how these requirements are balanced in response to injury is unknown. Here we demonstrate that after wounding or ultraviolet type B irradiation, melanocyte stem cells (McSCs) in the hair follicle exit the stem cell niche before their initial cell division, potentially depleting the pool of these cells. We also found that McSCs migrate to the epidermis in a melanocortin 1 receptor (Mc1r)-dependent manner and differentiate into functional epidermal melanocytes, providing a pigmented protective barrier against ultraviolet irradiation over the damaged skin. These findings provide an example in which stem cell differentiation due to injury takes precedence over stem cell maintenance and show the potential for developing therapies for skin pigmentation disorders by manipulating McSCs.

Figure 1

McSCs migrate from the hair follicle niche to the epidermis in response to injury or UVB irradiation. (a) X-gal–stained wound area of Trp2-LacZ mice before (0 d) and 5 or 8 d after wounding. (b) Pax3 (top) and H&E (bottom) staining of identical hair follicles at the indicated time points after wounding. UF, upper follicle; sg, sebaceous gland. Arrowhead indicates epidermal melanocyte. (c,d) Detection of melanocyte LRCs (arrowhead) in Tyr-rtTA;TetO-H2B-GFP mice^, at 0 d (c) and 4 d (d) after wounding. IFE, interfollicular epidermis. (e–g) BrdU- and Trp2-stained melanocytes before (e) and after (f,g) wounding and BrdU injection during the previous anagen phase. (h) Quantification of BrdU⁺Trp2⁺ melanocytes. The data is shown as the mean ± s.d. (i) Experimental scheme of the BrdU labeling of the melanocytes shown in e–g. In b and e, the insets show the white boxed areas at higher magnification. In a and c–g, the dashed lines indicate the epidermis-dermis border. Scale bars, 500 μm (a, top); 100 μm (a, bottom); 25 μm (b); 5 μm (insets in b); 20 μm (c–g); 10 μm (inset in e).

Figure 2

McSCs migrate directly from the hair follicle niche to the epidermis without proliferation. (a–c) Whole-mount images of the wound area and hair in the wound periphery of Trp2-LacZ mice at 12 d (a), 45 d (b) and 120 d (c) after wounding. The inset in b shows a bulge (arrowhead) in the unpigmented follicle stained with X-gal. (d) Quantification of the number of unpigmented hairs in the wound periphery. (e–g) Immunofluorescence for Trp2 and BrdU in skin from mice that were continuously injected with BrdU after wounding. (h) Immunofluorescence for K14, a marker for basal epidermal cells, Trp2 and BrdU. Insets show separate color images of the boxed area. (i) Schematic illustration of the results shown in e–h. (j) Quantification of BrdU⁻Trp2⁺ melanocytes in the wound periphery after 4 d. The dashed lines in b and e–g indicate the epidermis-dermis border. The data in d and j are shown as the mean ± s.d. Scale bars, 500 μm (a–c); 20 μm (eh and insets).

Figure 3

McSCs in Mc1R mutant mice show defects in migration to the epidermis. (a,b) X-gal–stained whole-mount and sectioned wound samples from Mc1r^e/e; Trp2-LacZ (a) and control Trp2-LacZ (b) mice after excisional wounding. (c) Number of Trp2⁺ epidermal melanocytes in the re-epithelialized area. (d) Quantification of BrdU⁻ epidermal melanocytes in the wound periphery after 5 d in Mc1r^e/e and wild-type control mice that were injected with BrdU after wounding. (e) Number of unpigmented hairs in the wound periphery after 30 d in Mc1r^e/e and wild-type control mice. (f) Trp2 staining of hair follicle bulbs from depilated Mc1r^e/e and wild-type control mice at 6 d after anagen induction. (g) Quantification of Trp2⁺ bulb melanocytes. (h) Schematic representation of events after excisional wounding in Mc1r^e/e and control mice. (i) Number of epidermal melanocytes from UVB-treated explant assays with skin from Trp2-LacZ control mice and Mc1r^e/e; Trp2-LacZ mice cultured with or without ACTH. (j) In vitro melanocyte migration assay with melan-A cells treated with or without ACTH. The data in c–e, g, i and j are shown as the mean ± s.d. NS, not statistically significant. *P < 0.05, **P < 0.01 determined by Student’s t test. Scale bars, 50 μm (a,b, top); 20 μm (a,b, bottom, f).

Figure 4

Epidermal melanocytes reconstitute McSCs in de novo hair follicles formed in the wound. (a) Epidermal view of the X-gal–stained wound area from wounded Trp2-LacZ mice (left) and an illustration of the location of epidermal melanocytes and the hair follicle neogenesis area (red dotted line) (right). (b,c) Views from the dermal side (b) and tissue sections (c) of neogenic follicles with Trp2⁺ melanocytes from wounded Trp2-LacZ mice. Dashed lines indicate outline of hair follicle. (d) Schematic illustration of melanocyte location during hair follicle neogenesis. (e) Whole-mount tyramide staining of wound epidermis at 22 d after excision showing pigment-producing melanocytes. (f) Quantification of unpigmented de novo hair follicles (HF) at 24 and 48 d after wounding. The data are shown as the mean ± s.d. (g) Whole-mount image of a pigmented neogenic hair follicle at 48 d after wounding (the second anagen of neogenic hair follicles). (h) Staining of neogenic hair follicles sections with Trp2 and the indicated melanocyte differentiation markers at 48 d after wounding. (i) Scheme of the ex vivo hair reconstitution assay. (j) Whole-mount (left) and tissue section (center) of X-gal–stained reconstituted hair follicles from nude mice that were injected with an epidermal cell suspension from UVB-irradiated Trp2-LacZ mice. Right, quantification of LacZ⁺ hair follicles in nude mice injected with cells from UVB-treated or non–UVB treated Trp2-LacZ mice. The data are shown as the mean ± s.d. The insets in e, g, h and j show magnified views of the boxed areas. Scale bars, 500 μm (a,e,g,j, left); 25 μm (b); 20 μm (c); 50 μm (h,j, center).