The chemokine SDF-1/CXCL12 regulates the migration of melanocyte progenitors in mouse hair follicles

Abstract

Mouse skin melanocytes originate from the neural crest and subsequently invade the epidermis and migrate into the hair follicles (HF) where they proliferate and differentiate. Here we demonstrate a role for the chemokine SDF-1/CXCL12 and its receptor CXCR4 in regulating the migration and positioning of melanoblasts during HF formation and cycling. CXCR4 expression by melanoblasts was upregulated during the anagen phase of the HF cycle. CXCR4-expressing cells in the HF also expressed the stem cell markers nestin and LEX, the neural crest marker SOX10 and the cell proliferation marker PCNA. SDF-1 was widely expressed along the path taken by migrating CXCR4-expressing cells in the outer root sheath (ORS), suggesting that SDF-1-mediated signaling might be required for the migration of CXCR4 cells. Skin sections from CXCR4-deficient mice, and skin explants treated with the CXCR4 antagonist AMD3100, contained melanoblasts abnormally concentrated in the epidermis, consistent with a defect in their migration. SDF-1 acted as a chemoattractant for FACS-sorted cells isolated from the anagen skin of CXCR4-EGFP transgenic mice in vitro, and AMD3100 inhibited the SDF-1-induced migratory response. Together, these data demonstrate an important role for SDF-1/CXCR4 signaling in directing the migration and positioning of melanoblasts in the HF.

Fig. 1

Expression patterns of SDF-1 and CXCR4 in the skin during mouse embryogenesis: Paraformaldehyde-fixed sections of skin harvested from E18 and E20 embryos showing SDF-1–EGFP (a) and CXCR4–EGFP (b,c) expression during hair follicle development at day E18 (a,b) and E20 (c). From E18 SDF-1–EGFP is expressed in the dermal cells enveloping the hair follicle (HF) and in the dermal papilla (DP) (a), whereas CXCR4–EGFP is expressed in a region that seems to correspond to the “Bulge” area (B) and in the DP (b). (c) shows GFP expression in the external epithelial layer of the hair follicle at E20. Arrows in (a,b) point to the dermal papilla. Insert in (b) shows a higher magnification ( × 2) of the area in box. Scale bar = 100 μm.

Fig. 2

Distribution and localization of melanoblasts in the embryonic skin of CXCR4 mutant mice: Paraformaldehyde-fixed sections of skin harvested from E18–20 embryos were immunostained for the melanoblast-specific marker tyrosinase-related protein-2 (TRP2) to show the distribution of melanoblasts and their localization in the skin of CXCR4+/+ and CXCR4−/− mice. Melanoblasts marked by TRP2 were regularly shaped and localized at about the same distance from the epidermis and the dermis in the HF in CXCR4+/+ mice (b,c), but irregularly positioned in the epidermis in CXCR4−/− mice (e,f). (a,d) are 4′-6-diamidino-2-phenylindole (DAPI) stained skin sections. (epidermis (ep); dermis (d); hair follicle (HF)). Scale bar = 50 μm.

Fig. 3

FACS purification of CXCR4-expressing cells: cells from embryonic skin of CXCR4–EGFP transgenic mice at E18–20 were isolated and sorted by fluorescence-activated cell sorting (FACS) based on EGFP fluorescence. (a) Sample FACS plot of the population of CXCR4–EGFP-expressing cells selected; CXCR4-expressing cells were selected as a highly fluorescent population (R3; right peak). (b–d) Epifluorescence (green) of dissociated embryonic skin cells before and after FACS purification co-stained for CXCR4 (red). (b) Mixed skin cells before FACS purification; only a very small percentage (1.83 ± 0.18%) of dissociated cells are CXCR4–EGFP-expressing cells that stain for CXCR4 receptors. (c) FACS purification results in an essentially pure population of EGFP-positive cells that all stained for CXCR4 receptors (c). (d) FACS-sorted EGFP-negative cells did not stain for CXCR4 receptors as shown by DAPI counter stain (blue, d). (e,f) Sample (Ca²⁺)i imaging plots of a population of FACS-sorted cells harvested from the skin of CXCR4–EGFP embryos (E18–20). In (e) CXCR4–EGFP-expressing cells responded to SDF-1, and ATP. SDF-1 and ATP-induced Ca responses were present in 100% of cells tested (n = 3 experiments). (f) EGFP-negative cells did not respond to SDF-1 but still responded to ATP. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

SDF-1 induces (Ca²⁺)i changes and acts as a chemoattractant for melanoblasts of embryonic skin: cells from embryonic skin of CXCR4–EGFP transgenic mice at E18–20 were isolated, sorted by fluorescence-activated cell sorting (FACS) based on EGFP fluorescence and subjected to immunocytochemistry for the melanoblast-specific marker TRP2 (a), (Ca²⁺)i imaging (b,c) and microchemotaxis (d) assays. (a) Merged image of CXCR4–EGFP expression (green) and immunostaining for TRP2 showing FACS-sorted EGFP cells all stained for TRP2. In (b) CXCR4–EGFP-expressing cells responded to stem cell factor (SCF) (84/95: 88%), and endothelin-3 (ET-3) (95/95: 100%), in addition to SDF-1 and ATP, indicating that they are melanoblasts. (c) EGFP-negative cells did not respond to SCF but still responded to ET-3 (< 10%) and ATP. (d) FACS-sorted EGFP-positive cells as characterized as in (a,b) migrated towards an SDF-1 (50 nM) gradient (120 min). The migration responses by FACS-sorted EGFP-positive cells towards SDF-1 was inhibited by the CXCR4 antagonist AMD3100 (10 μM) (n = 3), indicating that they were mediated by the CXCR4 receptor. *P<0.05, significantly different from controls. **P<0.05, significantly different from SDF-1-treated groups. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Expression patterns of CXCR4 within the postnatal skin of CXCR4–EGFP transgenic mice: Paraformaldehyde-fixed sections of skin illustrating CXCR4–EGFP expression during hair follicle cycling at postnatal days (P): P1, P7, P9, P12, P15, and P24, P27, P32 to cover the first and the second anagen (ana); P19, and P38 to cover the first and the second catagen (cata); and P21 and P45 for the first and the second telogen (telo), respectively. EGFP appeared to be strongly expressed in the dermal papilla (DP) and along an epithelial layer of the hair follicles (HFs) during the anagen phases of the HF cycles. At catagen (P19 and P38) and telogen (P21 and P45) HFs, only a small number of EGFP-positive cells appeared to be located at the base of the follicle epithelium. All panels (a–i) are of the same magnification (scale bar in panel (b) = 100 μm). Insert shows a higher magnification ( × 2) of the boxed area in (c) highlighting CXCR–EGFP-expressing cells probably representing cells of the bulge. Arrows in panels (f,g,j,k,i) point to CXCR–EGFP-expressing cells probably representing cells of the DP or the bulge. Outer root sheath (ORS); bulge region (B); dermal papilla (DP); anagen, (ana); catagen (cata); telogen (telo).

Fig. 6

CXCR4–EGFP-expressing cells are located in the HF bulge area and the outer root sheath: paraformaldehyde-fixed sections of skin harvested from CXCR4–EGFP mice at P7-9 illustrating immunostaining for CXCR4–GFP, K17 (marker of the outer root sheath (ORS)), K15 (marker of the HF bulge), and CXCR4. (a–c) are merged image of immunostaining for GFP (a) and K17 (b), showing CXCR4–EGFP expression in the ORS. (d–f) are merged image of immunostaining for GFP (a) and K15 (b), showing CXCR4–EGFP expression in the HF bulge area. Thus, CXCR4–EGFP appears to be expressed in the bulge area (B) and the ORS, regions known to be rich of HF stem/progenitor cells. (g,h) are merged images of EGFP expression (green) and immunostaining for CXCR4 (red) showing expression of CXCR4 by cells of an epithelial layer of the HF. (g, longitudinal view of a hair follicle and h, transverse view of hair follicles). Scale bar in panels (g,h) = 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Characterization of CXCR4-expressing cells: Cells from postnatal anagen skin of CXCR4–EGFP transgenic mice at P4–7 were isolated, sorted by fluorescence-activated cell sorting (FACS) based on EGFP fluorescence, and subjected to immunocytochemistry for nestin, Lex and the proliferation cell nuclear factor (PCNA), markers generally used for the identification of progenitor cells. (a–c), illustrates immunostaining of CXCR4–EGFP FACS-sorted cells for nestin (a), Lex (b) and PCNA (c), thus indicating that these cells express markers of progenitor cells.

Fig. 8

Expression patterns of nestin–EGFP in the postnatal skin of transgenic mice: Confocal images from paraformaldehyde-fixed sections of skin showing nestin–EGFP expression during hair follicle cycling at postnatal day 1, 2, 4, 7 and 26, 30, 33 to cover the first and the second anagen (ana); P19, and P37 to cover the first and the second catagen (cata); and P21 for the first telogen (telo). During anagen (P1, P2, P4, P7, P26, P30, P33) nestin–EGFP is expressed in the bulge area and the ORS, but not in the DP (in contrast to CXCR4–EGFP expression, Fig. 5), but only a small number of EGFP-positive cells appeared to be located at the base of the follicle epithelium at catagen HF at P19 and P37, and telogen HF at P21. Scale bar in panels (a–i) = 100 μm. Insert shows a higher magnification ( × 2) of the boxed area in (e) highlighting nestin–EGFP-expressing cells probably representing cells of the bulge area. anagen, (ana); catagen (cata); telogen (telo).

Fig. 9

Expression patterns of SDF-1 within the postnatal skin of SDF-1 transgenic mice: Confocal images from paraformaldehyde-fixed sections of skin showing SDF-1–EGFP expression during hair follicle cycling at postnatal days (P): P1, P7, P11, P14 and P24, P28, P33, P35 to cover the first and the second anagen (ana); P19, and P38 to cover the first and the second catagen (cata); and P21 and P41, P44, P46, P50 for the first and the second telogen (telo). (a–p) SDF-1–EGFP is abundantly expressed in dermal cells and in the DP forming a gradient throughout the postnatal HF cycle at anagen, catagen and telogen phases. In (q–s) we used BAC transgenic mice (SDF-1–mRFP1 mice), which expressed an SDF-1–mRFP fusion protein crossed with CXCR4–EGFP mice to generate a new line of mice expressing both CXCR4–EGFP and SDF-1–mRFP. Confocal images of sections of skin from these mice demonstrate the relative localization of CXCR4–EGFP-expressing cells in anagen HFs (green in q and s) and that of SDF-1–mRFP (red in r and s) showing the production and localization of SDF-1–mRFP protein along the path of CXCR4-expressing cells. Scale bar in panels (a–k,o–p) = 100 μm, (l–n,q–s) = 50 um. anagen, (ana); catagen (cata); telogen (telo). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 10

SDF-1 induces (Ca²⁺)i changes and acts as a chemoattractant for CXCR4–EGFP-expressing cells of postnatal skin: CXCR4–EGFP-expressing cells harvested from anagen skin at P4–7 and isolated by FACS migrated towards an SDF-1 (50 nM) gradient (120 min) and this was inhibited by the CXCR4 antagonist AMD3100 (20 μM) (n = 4) (a). Consistent with the fact that most of the CXCR4–EGFP-expressing cells stained for nestin, FACS–EGFP-expressing cells harvested from anagen skin of nestin–EGFP mice at P4–7 also responded to SDF-1, and ATP (n = 3) (b) in the Ca response assay. Similarly, nestin–EGFP-expressing cells migrated towards an SDF-1 (50 nM) gradient and this was inhibited by AMD3100 (20 μM) (n = 2) (c). *P<0.05, Significantly different from respective controls in a and c. **P<0.05, significantly different from respective SDF-1-treated groups in a and c.

Fig. 11

Expression of SOX10, a neural crest marker, by CXCR4–EGFP-expressing cells: Skin sections and FACS-sorted skin cells harvested from an anagen postnatal day P7 of CXCR4–EGFP mice were subjected to in situ hybridization with a probe specific for SOX10, using digoxygenin (DIG) labeling (a,b,e) and fluorescence in situ hybridization (FISH) performed together with immunostaining with an EGFP antibody (c,d,f). (a,b,e) show SOX10 mRNA expression by hair follicles along the outer root sheath (ORS) and the basal layer of the epidermis (a) and by CXCR4–EGFP FACS-sorted cells (e); (b) negative control for SOX10 sense riboprobe. (c,d,f) are merged images of CXCR4–EGFP expression immunostained with EGFP antibody and images processed with FISH to show the colocalization of SOX10 and CXCR4–EGFP . Arrows in c and d, point to the ORS in a longitudinal view of a hair follicle (c), and transverse view of hair follicles (d). Arrowheads in c and d point to the absence of SOX10 staining in the HF matrix. (f) shows FISH for SOX10 and CXCR4–EGFP in P7-CXCR4–EGFP FACS-sorted cells. Scale bar in panels (a–d) = 50 μm, (e,f) = 20 um.

Fig. 12

Expression of the melanocyte-specific marker tyrosinase-related protein 2 (TRP2) by CXCR4–EGFP-expressing cells: The expression of TRP2 was carried out using immunohistochemistry on skin sections from P7-CXCR4–EGFP transgenic mice. The overlap of CXCR4–EGFP cells (green) and Alexa 633-labeled TRP2 cells (red) shows that most of the CXCR4–EGFP cells expressed the melanoblast-specific marker TRP2 (a,b) (a, longitudinal view of a hair follicle, (b) transverse view of hair follicles. In (c) immunostaining for the ORS keratinocyte marker K14 was carried out on skin sections in P7-CXCR4–EGFP transgenic mice to show the expression of K14 relative to CXCR4–EGFP cells and observed that few cells expressed the ORS keratinocyte marker K14 (arrow in c). (d–f) immunostaining for TRP2 on P7-CXCR4–EGFP FACS-sorted cells. (d) Merged image of immunostaining for the melanoblast marker TRP2 on mixed skin cells before FACS purification showing only a very small percentage (<2%) of cells that are EGFP-positive (green, arrows in d) and which stain for TRP2 (red, arrows in d). In (e) Most EGFP-positive cells stained for TRP2. (f) EGFP-negative cells as shown by DAPI counterstain (blue) did not stain for TRP2. Scale bar in panels (a–c) = 50 μm and (c) = 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 13

CXCR4 expression is undetectable in differentiated melanocytes in vitro and in vivo: (a–b) Confocal images of immunostaining for CXCR4 (a) and for the differentiated melanocyte marker tyrosinase-related protein 1 (TRP1) (b) in differentiated melanocytes. In this experiment, CXCR4–EGFP FACS-sorted TRP2 positive cells were cultured with bFGF (2.5 ng/ml), endothelin-3 (100 μM) and dibutyryl adenosine cAMP (0.5 mM) or SCF (2 ng/ml) for 15 days to induce differentiation to melanocytes. After 15 days, most of the cells no longer exhibited CXCR4–EGFP fluorescence or staining with CXCR4 antibody (a), became positive for the melanocyte marker TRP1 (b), but did not respond to SDF-1 (c) in the SDF-1-induced (Ca²⁺)i imaging assay. (d–f) are merged images of EGFP fluorescence (green) with TRP1 fluorescent immunostaining (red) on anagen skin sections from P7-CXCR4–EGFP (d), P30 nestin–EGFP (e) and P7-SDF–EGFP (f). Arrows in (d) point to cells in close contact with and around the dermal papilla (DP) that expressed both CXCR4 and the melanocyte marker TRP1. This was confirmed in merged images of SDF-1–EGFP with TRP1 (arrows in f). In contrast, TRP1+ positive cells not in direct contact with the DP and located in the upper part of the matrix bulb did not appear to exhibit CXCR4–EGFP fluorescence (arrowheads in d–f). Scale bar = 50 μm.

Fig. 14

Migration and localization of melanoblasts in skin explant preparations is disrupted by AMD3100, a selective antagonist for CXCR4 receptors: Skin explant cultures prepared from E13.5 mouse embryos were treated with the CXCR4 antagonist AMD3100 (100 μM) for 48 h and assessed for the melanoblast marker tyrosinase-related protein 2 (TRP2) immunoreactivity after 10 days in culture. Immunostaining for TRP2 showed that most melanoblasts positioned in the dermis (d) in control explants (arrows, a) but accumulated in the epidermis (ep) in AMD3100-treated explants (arrows, b). Scale bar = 100 μm.