Maintenance of distinct melanocyte populations in the interfollicular epidermis

Abstract

Hair follicles and sweat glands are recognized as reservoirs of melanocyte stem cells (MSCs). Unlike differentiated melanocytes, undifferentiated MSCs do not produce melanin. They serve as a source of differentiated melanocytes for the hair follicle and contribute to the interfollicular epidermis upon wounding, exposure to ultraviolet irradiation or in remission from vitiligo, where repigmentation often spreads outwards from the hair follicles. It is unknown whether these observations reflect the normal homoeostatic mechanism of melanocyte renewal or whether unperturbed interfollicular epidermis can maintain a melanocyte population that is independent of the skin's appendages. Here, we show that mouse tail skin lacking appendages does maintain a stable melanocyte number, including a low frequency of amelanotic melanocytes, into adult life. Furthermore, we show that actively cycling differentiated melanocytes are present in postnatal skin, indicating that amelanotic melanocytes are not uniquely relied on for melanocyte homoeostasis.

Keywords: epidermis; hair follicle; interfollicular; melanocyte; stem cell.

Figure 1

Functional melanocytes persist in the absence of hair follicles. (A) Detection of melanin by Masson‐Fontana stain in epidermis and dermis of wild‐type (Eda ^+/Y) and mutant (Eda ^Ta/Y) tail skin at 10 weeks. Scale bar 50 μm. HF denotes hair follicles and associated sebaceous glands. (B) Detection of active melanocytes by L‐DOPA staining in wild‐type and mutant epidermis at 16 weeks. Scale bar 50 μm. (C) Quantification of melanocyte density in all tail interfollicular epidermis and specifically in the scale regions through adult life in wild‐type (WT) and mutant (Ta/Y) mice. A stable melanocyte population is maintained in the mutant in the absence of hair follicles. Error bars represent SEM. Significant difference was calculated using a Student's t test * P < 0.05, ** P < 0.01. (D) Distribution of melanocytes in 4‐week‐old wild‐type and Eda ^Ta/Y epidermis, revealed by X‐gal staining (blue colour) of Dct::lacZ transgenic tissue. Scale bar 0.5 mm.

Figure 2

Amelanotic melanocytes exist in the interfollicular epidermis. (A) Isolated epidermis from Tyr::Cre; Rosa26::mT/mG at 16 weeks. Melanocytes express membrane‐EGFP, while other cells have membrane‐tdTomato. Left panel, fluorescence in red and green channels. Right panel, fluorescence merged with brightfield. Hair follicles have melanocyte populations at the bulb (Bb) and bulge (Bg). Scale bar 200 μm. (B) L‐DOPA‐negative interfollicular epidermal melanocytes (indicated by arrows) are interspersed amongst the differentiated melanocytes in wild‐type (Eda ^+/Y) and mutant (Eda ^Ta/Y) isolated epidermis. Scale bar 25 μm. (C, D) L‐DOPA‐negative melanocyte density in 16‐week‐old wild‐type and mutant interfollicular epidermis, by area (C) and as a percentage of total melanocytes (D). Error bars represent SEM.

Figure 3

Melanin‐producing melanocytes are capable of cell division in mouse skin. (A) Schematic showing marking of cell cycle phase using the Fucci2a system. (B) Differentiated, melanin‐containing melanocytes in 9‐day‐old wild‐type mice carrying the Tyr::Cre transgene and R26Fucci2aR allele are found in the S/G2/M phases of the cell cycle (arrow). Scale bar 50 μm. (C) Panels show time lapse of explant cultured Tyr::Cre; R26Fucci2aR skin at P19. Melanocytes in S/G2/M phases of the cell cycle (green fluorescent signal, indicated by circles) undergo division (arrows) without losing melanin pigment. Scale bar 50 μm.