ARTICLE |
Correspondence to: Ralf Paus, Dept. of Dermatology, University Hospital Eppendorf, University of Hamburg, Martinistr. 52, D-20246 Hamburg, Germany. E-mail: paus@uke.uni-hamburg.de
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Summary |
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Disruption of the c-Kit/stem cell factor (SCF) signaling pathway interferes with the survival, migration, and differentiation of melanocytes during generation of the hair follicle pigmentary unit. We examined c-Kit, SCF, and S100 (a marker for precursor melanocytic cells) expression, as well as melanoblast/melanocyte ultrastructure, in perinatal C57BL/6 mouse skin. Before the onset of hair bulb melanogenesis (i.e., stages 04 of hair follicle morphogenesis), strong c-Kit immunoreactivity (IR) was seen in selected non-melanogenic cells in the developing hair placode and hair plug. Many of these cells were S100-IR and were ultrastructurally identified as melanoblasts with migratory appearance. During the subsequent stages (5 and 6), increasingly dendritic c-Kit-IR cells successively invaded the hair bulb, while S100-IR gradually disappeared from these cells. Towards the completion of hair follicle morphogenesis (stages 7 and 8), several distinct follicular melanocytic cell populations could be defined and consisted broadly of (a) undifferentiated, non-pigmented c-Kit-negative melanoblasts in the outer root sheath and bulge and (b) highly differentiated melanocytes adjacent to the hair follicle dermal papilla above Auber's line. Widespread epithelial SCF-IR was seen throughout hair follicle morphogenesis. These findings suggest that melanoblasts express c-Kit as a prerequisite for migration into the SCF-supplying hair follicle epithelium. In addition, differentiated c-Kit-IR melanocytes target the bulb, while non-c-Kit-IR melanoblasts invade the outer root sheath and bulge in fully developed hair follicles. (J Histochem Cytochem 50:751766, 2002)
Key Words: hair follicle morphogenesis, c-Kit, stem cell factor, S100, C57BL/6 mouse, melanoblast, melanocyte, immunohistochemistry, ultrastructure
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Introduction |
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DURING embryonic and fetal development signaling of the receptor tyrosine kinase c-Kit [mapped to the white-spotting (W) locus in mice] and its cognate ligand stem cell factor [SCF, also known as mast cell growth factor, steel factor; mapped to the steel (SL) locus in mice] is important for melanocyte migration from the neural crest to target tissues (
In the mouse, at least 90 loci have been identified that affect coat color (Mouse Genome Database, http://www.informatics.jax.org/, 1998). Among these are the widely known mutations in the W and SL loci. Our understanding of the role of c-Kit/SCF signaling in differentiation, proliferation, and migration of melanocyte precursors is derived in large part from the analysis of spontaneous or experimentally induced mutations at these two loci (
Studies of melanoblast/melanocyte regulation through c-Kit/SCF signaling have focused mainly on the phenotypic description of pigmentation effects after c-Kit/SCF signal disruption by mutation or antibody treatment (
Interestingly, c-Kit is strongly expressed on putative melanoblasts in the developing hair follicle epithelium (
The temporally restricted c-Kit/SCF expression in the adult hair pigmentary unit prompted us to investigate whether, first, c-Kit and SCF expression correlated with melanocyte migration and differentiation during the establishment of the hair pigmentary unit in hair follicle development in C57BL/6 mice and, second, whether c-Kit expression is lost from specific melanocyte subpopulations in the fully developed hair follicle.
The C57BL/6 mouse model for hair research enables the study of c-Kit/SCF expression in a physiological, developmentally regulated epithelialneuroectodermalmesenchymal interaction system (
For the current study we employed specific immunohistochemistry (IHC) to detect c-Kit on epidermal and follicular melanoblasts and melanocytes and SCF in epithelial cells. This was complemented by the assessment of S100-IR to detect melanocytic precursor cells within the hair follicle, because this marker can be used to detect melanogenic precursor neural crest cells (
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Materials and Methods |
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Animals and Tissue Collection
C57BL/6 breeding pairs were purchased from Charles River (Sulzfeld, Germany). The mice were housed with 12-hr light periods and were fed water and mouse chow ad libitum. As described below, E16 and E18 fetuses were obtained by Cesarian section from time-mated mice sacrificed on the respective day of gestation. E1 was counted as the day of mating. Birth usually occurred on E19, which was considered postnatal day 1 (P1). Skin samples of neonatal mice were harvested on P1, P3, P5, and P8 to trace migrating and differentiating melanocytes during the development of fetal and neonatal back skin and pelage hair follicles. Skin samples were snap-frozen in liquid nitrogen and further processed for immunohistochemistry as described previously (
Immunohistochemistry
Acetone-fixed cryostat sections (7 µm) were stained by IHC for c-Kit and SCF for either analysis by brightfield or immunofluorescence microscopy. Briefly, for brightfield light microscopy, immunolabeling of c-Kit (CD 117; rat anti-mouse polyclonal antibody, 1:200; Pharmingen, San Diego, CA) and SCF (rabbit anti-mouse polyclonal antibody, 1:20; Genzyme, Cambridge, MA) was performed according to our previously published protocols (c.f.
For immunofluorescence staining of S100, sections were incubated with the primary antibody (S100; rabbit anti-bovine polyclonal antibody 1:500; Sigma, St Louis, MO) (
For immunofluorescence staining of c-Kit or SCF, tyramide amplification was performed after incubation with the primary antibodies to c-Kit (CD 117; rat anti-mouse polyclonal antibody 1:1000; Pharmingen, San Diego, CA) (
For double staining of S100 and c-Kit, tissue sections were first incubated with anti-c-Kit primary antibody overnight, followed by tyramide amplification, and then incubated with anti-S100 following the above protocol using fluorescein isothiocyanate (FITC)-conjugated F(ab)2 fragments of goat anti-rabbit IgG as described before (
Mast cells were counterstained with fluorescein-labeled avidin according to our previously published protocol (
Sections were examined at x400 magnification using a Zeiss Axioscope microscope. The appearance of IR cells in defined skin compartments was assessed as previously described in detail (
HRLM and TEM
Selected hair follicle epithelial compartments that exhibited strong c-Kit-IR by brightfield microscopy using 7-µm frozen sections (i.e., the early budding hair follicle epithelium, the developing outer root sheath, and the developing proximal hair bulb) were further screened by HRLM (0.51-µm resin sections) and by TEM for the presence of cells with ultrastructural features of melanoblasts and melanocytes (
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Results |
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c-Kit-IR Highlights Selected Non-dendritic Epidermal Cells Before and During Early Hair Follicle Development
Approximately 10% of cells in the epidermis exhibited c-Kit-IR. These cells were non-dendritic and were located in the basal and suprabasal layers of the epidermis before and during the earliest stages of hair follicle development, i.e., before formation of an inner root sheath (stage 0 to 3) (Fig 1A0). From the day of birth (P1), c-Kit-IR epidermal cells became less abundant and were increasingly concentrated in the basal layer of the epidermis (Fig 2A42A6). By the time the majority of hair shafts penetrated the epidermis (stage 7 to 8, P3) only a few isolated c-Kit-IR cells were detectable in the epidermis (not shown).
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By contrast, all cells in the epidermis expressed S100-IR throughout hair follicle development. Clearly, therefore, S100-IR was not a suitable melanoblast or melanocyte marker within the epidermis (Fig 1B01B6 and 2B02B6) (
HRLM, however, revealed the presence of isolated, "clear cells" with morphological features of melanoblasts (e.g., round cells with clear cytoplasm and heterochromatic nuclei). These cells were located in the basal layer of the epidermal compartment before and throughout hair follicle development (Fig 1C0 and 1C12).
Isolated cells with morphological features of melanoblasts were also observed by EM in the epidermis before the onset of morphologically appreciable hair follicle development. Again, these cells were distinguishable from neighboring epithelial cells by their clearer cytoplasm and their lack of cytokeratin filaments or desmosomes. Rarely, (pre)melanosomes or abundant Golgi and endoplasmic reticulum were detectable in these non-dendritic cells (Fig 1D0a and 1D0b). This low level of cellular activity in intraepidermal melanoblasts was also indicated by heterochromatic nuclei, which contrasted markedly with the metabolically highly active proliferating and euchromatic keratinocytes nearby.
c-Kit-IR Highlights Dendritic Cells with Melanocytic Features in the Developing Hair Follicle Epithelium Before the Onset of Follicular Pigment Production
During the early stages of hair follicle development (i.e., stages 13/4), individual c-Kit-IR cells with few dendrites were observed to increasingly invade the hair follicle epithelium (Fig 1A11A4 and 2A12A4). Still close to the epidermis but now located in the center of the developing hair follicle epithelium, these cells had a suggestive migratory appearance, as evidenced by their mono- to tripolar shape with longish oval nuclei oriented parallel to the growth direction of the highly proliferative hair follicle epithelium (
In contrast to the ubiquitous and homogeneous epidermal S100 staining, the hair follicle epithelium was strikingly S100-IR negative. However, within this S100-IR negative epithelium, individual mono- or bipolar dendritic S100-IR cells were detected, preferentially distributed in the center of the hair follicle epithelium (Fig 1B11B4 and 2B12B4) or occasionally in the region just above the dermal papilla (Fig 1B3). Interestingly, immunoreactivity was reduced in S100-IR cells in contact with the basement membrane separating hair matrix from dermal papilla. At this stage of hair follicle morphogenesis, S100-IR cells were fewer than c-Kit-IR cells.
With HRLM and EM, melanoblasts containing the occasional (pre)melanosome were detected in similar hair follicle compartments to those containing c-Kit- and S100-IR cells, i.e., the center of the hair follicle epithelium at stage 24 (Fig 1C21C4, 1D2a1D4b and 2C22C4 and 1D2a1D4b). Frequently, the direction of melanoblast migration could be inferred by cell orientation, distribution of the nucleus within the advancing portion of the cell, and by the presence of trailing cytoplasm. These features suggested that the direction of cell migration was away from the epidermis and towards the dermal papilla. There was also evidence that the hair follicle's complement of melanocytes was derived in part from spatially restricted proliferation of melanoblasts in the hair follicle epithelium during stage 12 of hair follicle morphogenesis (Fig 1D1 and 1D2).
c-Kit-IR Melanocytes Contribute to the Establishment of the Hair Follicle Pigmentary Unit
Hair bulb melanogenesis was first seen in the bulbar melanocyte subpopulation during stage 4/5 to 6 of hair follicle morphogenesis (
S100-IR continued to be expressed in single bipolar cells with a migratory appearance located in the center of the hair follicle epithelium (Fig 2B5 and 2B6), and also occasionally within the pigmenting hair bulb just above the dermal papilla.
HRLM and TEM confirmed the above findings by clearly showing melanogenically active and polydendritic melanocytes in the hair bulb (Fig 2C5, 2C6a, 2D5, and 2D6). In addition, non-melanogenic melanoblast-like cells were detected in the outer root sheath near the bulge region (Fig 2C6b).
At the End of Hair Follicle Morphogenesis c-Kit-IR Cells Disappear from the ORS and Bulge Region to Home into the Hair Follicle Bulb
c-Kit-IR cells were detected in the outer root sheath and bulge even in stage 7 hair follicles, just before the formation of the hair canal that permits exiting of the hair shaft to the skin surface (Fig 3A7a and 3A7b). However, c-Kit-IR cells were no longer detected in these locations on completion of hair follicle development in stage 8 (Fig 3, scheme 8). Instead, a large c-Kit-IR cell population displaying an epithelial cell phenotype, i.e., cobblestone-like IR pattern, became concentrated below Auber's line (Fig 3A7c, 3A7d, and 3A8). Moreover, c-Kit-IR in the melanogenic zone above and around the tip of the dermal papilla was comparatively weak and became increasingly obscured by the melanin that was produced in this region (Fig 3A8). However, the tyramide amplification method revealed two distinct c-Kit-IR cell populations within the hair bulb. One was a population of strongly c-Kit-IR melanocytes located in the melanogenic zone above/around the upper dermal papilla and the other located below Auber's line (Fig 4). Both groups were separated by a group of c-Kit-negative epithelial cells (Fig 4).
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S100-IR cells were no longer detected in the hair follicle epithelium of stage 7/8 hair follicles. Instead, this antigen was strongly expressed by Schwann cells in dermal and subcutaneous nerve fiber bundles in addition to hair follicle dermal papilla fibroblasts (Fig 3B7a and 3B7b) (
HRLM and TEM analysis not only demonstrated that melanocytes were located in the melanogenic zone above and around the upper dermal papilla (Fig 3C7a3C7f, 3D7a, and 3D7b) but additionally demonstrated that melanoblasts were distributed throughout the full length of the ORS of stage 7 hair follicles. Most intriguingly, c-Kit-IR cells could not be detected in the outer root sheath in stage 8 hair follicles. Significant cellular metabolic activity, including melanosome production, was apparent only in the melanocytes located in the hair follicle melanogenic zone, as evidenced by their dendricity, high level of melanogenesis, and euchromatic nuclei indicative of high levels of gene transcription (Fig 3C8a, 3C8b, 3D8a, and 3D8b). Melanoblast-like cells continued to be detectable in the fully developed stage 8 hair follicle, and resided mostly in the distal third of the outer root sheath, i.e., the isthmus and bulge region (Fig 3C8c, 3C8d, 3Dc, and 3Dd). No melanocytic cells could be demonstrated in the c-Kit-IR cell population below Auber's line (Fig 3C8a, 3C8b, 3D8a, and 3D8b).
c-Kit-IR Co-localizes with S100-IR in Selected Follicular Cells During the Early Stages of Hair Follicle Development
Double labeling of S100 and c-Kit using the combined immunofluorescence and tyramide amplification method allowed us to clearly demonstrate double stained cells with a migratory appearance within the hair follicle epithelium (Fig 5). When compared to our ultrastructural findings (Fig 1C2, 1D2a, 1D2b), these cells correlated with the location and morphological features of early melanoblasts. All S100-IR cells within the hair follicle epithelium of stage 16 hair follicles were also c-Kit-IR, but only a few c-Kit-IR cells were also S100-IR. Because the ABC method provided better anatomic orientation, we chose to display c-Kit-IR as brightfield light microscopic mono stainings rather than as fluorescent double stainings. However, the fluorescence method allowed better discrimination of single IR cells and fine-caliber structures such as dendrites or Schwann cells. Therefore, S100-IR was demarcated by immunofluorescence.
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Just before and during hair follicle development, an occasional melanocytic cell was detected in the dermal compartment by EM (not shown). However, despite the numerous c-Kit-IR cells within this compartment (Fig 2A32A8 and 5A35A8), no S100-IR isolated cells could be detected. In addition, double staining of S100 with fluorescein-labeled avidin, used as a marker for cutaneous mast cells (
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SCF-IR Is Distributed in the Hair Follicle Epithelium During Hair Follicle Morphogenesis
SCF-IR keratinocytes were located in the basal layer of the epidermis and ubiquitously in the hair follicle epithelium throughout hair follicle development. This distribution pattern was apparent from brightfield and tyramide amplification immunofluorescence microscopy (Fig 6). Before the development of an inner root sheath (i.e. stage 3), hair follicle epithelial cells were strongly SCF-IR (Fig 6A2 and 6A3). Similarly, hair matrix keratinocytes at the onset of hair shaft and pigment production were also strongly SCF-IR (Fig 6A5 and 6B5) although SCF-IR was weak in the developing inner root sheath (Fig 6A4 and 6B5). SCF was also strongly expressed in the outer root sheath keratinocytes, and in the hair bulb region that contains the hair follicle pigmentary unit in fully developed hair follicles (Fig 6A8, 6B5, and 6B8).
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Discussion |
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This IHC and ultrastructural study provides evidence that the majority of intraepithelial c-Kit-IR cells present in developing murine back skin are melanoblasts undergoing migration into the developing hair follicle epithelium. Melanocytes that migrate into the follicular pigmentary unit retain c-Kit-IR, whereas those melanocytes/melanoblasts that remain in the outer root sheath lose c-Kit IR on completion of hair follicle development. Our study reveals a number of interesting additional features of the melanocytic cells detected: (a) Within the epidermis, the majority of c-Kit-IR putative melanoblasts are difficult to detect and are ultrastructurally undifferentiated. (b) Within the hair follicle epithelium, c-Kit-IR cells that exhibit a migratory appearance also express S100, a marker for melanocytic neural crest-derived cells (
These observations enable us to speculate on a role for c-Kit/SCF signaling in melanoblast migration on the one hand, and in melanocyte differentiation and maintenance on the other hand. In this context, two issues deserve special consideration: the striking developmental regulation of the expression patterns and the functional significance of c-Kit/SCF signaling for skin and hair biology.
The developmentally regulated establishment of the neonatal hair follicle pigmentary unit in C57BL/6 mice is an ideally suited model to dissect the exact contributions of c-Kit/SCF signaling to the choreography of melanoblast migration and differentiation in normal murine skin in vivo. In this model, it is easy to appreciate, even by light-microscopy, the onset of follicular pigment production, although whether or not each intraepidermal and intrafollicular c-Kit-IR cell represents a melanoblast or melanocyte during hair follicle morphogenesis can be clarified definitively only by immuno-EM double-labeling techniques applied to ultrathin sections of precisely targeted intraepithelial follicle compartments (unpublished data). However, the only hair follicle cell subpopulations that lack epithelial specialization by ultrastructural analysis include melanocytes, intraepithelial T-cells (DETC), and Langerhans cells (
One group of c-Kit-IR cells within the fully developed hair bulb deserves special attention. This group of cells is located proximal to Auber's line. They do not display melanocytic features and, given their sheer numbers, are most probably epithelial. However, the analysis of a potential role for c-Kit/SCF signaling within the hair follicle epithelium was beyond the scope of the current study (unpublished data; and
Most interestingly, the S100 antibody employed here to detect melanocytic cells of neural crest origin (
The ubiquitous distribution of SCF in the epithelial compartment suggests that it is unlikely to provide a chemoattractant gradient to guide melanoblast migration into and within the developing hair follicle epithelium. However, sufficient levels of SCF in the basolateral cell compartment of epidermal and follicular keratinocytes may be necessary to promote migration and differentiation of c-Kit-expressing melanoblasts within the designated hair follicle target compartments. It is interesting to note that SCF can exist in skin as a membrane-bound and a soluble form (
Correct migration and differentiation of melanoblasts within the hair follicle epithelium appears to require the localization of membrane-bound SCF to the basolateral cell compartment (
Unfortunately, the techniques employed in this study did not enable us to differentiate between membrane-bound and soluble SCF. In addition, homing of c-Kit-IR melanoblasts may require chemoattraction provided by the co-signaling of the c-Kit/SCF signaling system with growth factors such as endothelin (
Mouse experiments employing c-Kit neutralizing antibodies provide additional functional evidence for the important role of c-Kit/SCF signaling in survival and migration of melanoblasts, not only from the neural crest to the skin but also within developing hair follicles. When c-Kit-neutralizing antibodies are injected just before hair follicle development, i.e., before migration of melanoblasts from the epidermis into developing hair follicles, the entire coat remains white (
These findings concur with our understanding of the development of coat color in successive waves of hair follicle generations (
This study therefore provides an easy-to-use guide through the ultrastructural appearance and distribution of melanoblasts in the light of their c-Kit expression throughout hair follicle morphogenesis. Moreover, it provides suggestive evidence for an important role of the c-Kit/SCF signaling system in the development and maintenance of active bulbar pigment production in the growing hair follicle. Future co-localization experiments, e.g., with the aid of immuno-EM double stainings and the analysis of c-Kit regulation during adult hair follicle cycling, will provide further insight into the timing and mechanisms that regulate the c-Kit-/c-Kit+ switch between "passive" and "active" follicular melanocytes. This knowledge may be useful in the treatment of pigmentation disorders. For example, recombinant SCF has been used in clinical trials for the mobilization of stem cells in chemotherapy-treated hematologyoncology patients. Few side effects were experienced other than a flare and wheal reaction and focal hyperpigmentation at the site of subcutaneous injection (
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Acknowledgments |
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Supported in part by a grant from the Deutsche Forschungsgemeinschaft (DFG Pa 345/6-2) and by Cutech Srl., Venice, to R. P.
Received for publication June 5, 2001; accepted December 19, 2001.
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