Journal of Histochemistry and Cytochemistry, Vol. 50, 751-766, June 2002, Copyright © 2002, The Histochemical Society, Inc.


ARTICLE

Migration of Melanoblasts into the Developing Murine Hair Follicle Is Accompanied by Transient c-Kit Expression

Eva M. J. Petersa,b, Desmond J. Tobinc, Natasha Botchkarevaa, Marcus Maurerd, and Ralf Pausa
a Department of Dermatology, University Hospital Eppendorf, University of Hamburg, Germany
b Chamlé, Campus Virchow Clinic, Berlin, Germany
c Department of Biomedical Sciences, University of Bradford, Bradford, United Kingdom
d Department of Dermatology, University Hospital Johannes Gutenberg–University, Mainz, Germany

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


  Summary
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Summary
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Materials and Methods
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Discussion
Literature Cited

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 0–4 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 50:751–766, 2002)

Key Words: hair follicle morphogenesis, c-Kit, stem cell factor, S100, C57BL/6 mouse, melanoblast, melanocyte, immunohistochemistry, ultrastructure


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Motro et al. 1991 ; Nishikawa et al. 1991 ; Kunisada et al. 1998b ). c-Kit is expressed on melanoblasts from the time they leave the neural crest and continues to be expressed on melanocytic cells of postnatal animals, while its ligand SCF is expressed in their microenvironment (Motro et al. 1991 ; Nishikawa et al. 1991 ; Kunisada et al. 1998b ).

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 (Jackson 1994 ). On the basis of these analyses, epithelium-derived SCF is now generally regarded as an important regulator of c-Kit-expressing melanoblasts and melanocytes in mammalian skin, where it modulates melanocyte migration (Grabbe et al. 1994 ; Scott et al. 1994 ; Wehrle-Haller and Weston 1999 ; Botchkareva et al. 2001 ), differentiation (Lahav et al. 1994 ; Luo et al. 1995 ), melanogenesis (Luo et al. 1995 ; Costa et al. 1996 ), and survival/apoptosis (Ito et al. 1999 ).

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 (Brannan et al. 1991 ; Wehrle-Haller and Weston 1995 , Wehrle-Haller and Weston 1999 ; Yoshida et al. 1996 ). Alternately, these have analyzed proliferation, differentiation, and apoptosis in c-Kit-expressing melanoblasts after SCF treatment in culture (Ito et al. 1999 ; Jordan and Jackson 2000 ). Thus, lacking in previous studies have been a detailed account of when and where melanoblasts actually express c-Kit during hair follicle morphogenesis and whether SCF expression is differentially regulated as the hair follicle pigmentary unit is being constituted during hair follicle development.

Interestingly, c-Kit is strongly expressed on putative melanoblasts in the developing hair follicle epithelium (Jordan and Jackson 2000 ), whereas only some follicular melanocytes in adult cycling hair follicles appear to express c-Kit (Grichnik et al. 1996 ; Botchkareva et al. 2001 ).

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 epithelial–neuroectodermal–mesenchymal interaction system (Slominski and Paus 1993 ; Slominski et al. 1993 ; Maurer et al. 1995 ; Maurer et al. 1997 ; Paus et al. 1997 , Paus et al. 1999 ). Melanogenesis in the hair bulb is first detected in stage 4/5 hair follicles with the formation of a pigmented hair shaft and continues through to stage 8, when the hair shaft penetrates the surface epidermis (Paus et al. 1999 ). After a defined period of sustained growth, the hair follicle enters a short phase of rapid organ involution (catagen), thereby initiating the first hair cycle. Catagen is followed by a short period of relative resting (telogen) until the first wave of active hair growth (anagen) is induced again (Paus et al. 1999 ; Muller-Rover et al. 2001 ).

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 (Ito et al. 1993 ). In addition, high-resolution light microscopy (HRLM) and transmission electron microscopy (TEM) (Tobin et al. 1998 , Tobin et al. 1999 ) were conducted on selected epithelial tissue compartments of the hair follicle to define intrafollicular melanocytic cell populations ultrastructurally.


  Materials and Methods
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Summary
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Materials and Methods
Results
Discussion
Literature Cited

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 (Hoffman et al. 1996 ; Paus et al. 1999 ).

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. Eichmuller et al. 1996 , Eichmuller et al. 1998 ; Paus et al. 1998 ) employing the ABC technique (Vectastain Elite Kit; Vector, Burlingame, CA). Nuclei were counterstained with hematoxylin.

For immunofluorescence staining of S100, sections were incubated with the primary antibody (S100; rabbit anti-bovine polyclonal antibody 1:500; Sigma, St Louis, MO) (Botchkarev et al. 1997a ) overnight at room temperature (RT) in a humidified chamber, followed by incubation for 1 hr at 37C with tetramethyl rhodamine-isothiocyanate (TRITC)-conjugated F(ab)2 fragments of goat anti-rabbit IgG (Jackson Immunoresearch; West Grove, PA) at dilution 1:200.

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) (Schindler and Roth 1996 ; van Gijlswijk et al. 1997 ) or SCF (rat anti-recombinant mouse SCF monoclonal antibody 1:1000; R&D Systems, Minneapolis, MN) (Schindler and Roth 1996 ; van Gijlswijk et al. 1997 ) according to the manufacturer's instructions [Renaissance TSA Direct (Red); NEN Life Science Products, Boston, MA] and previously published protocols (Botchkareva et al. 2001 ).

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 (Botchkarev et al. 1997a ).

Mast cells were counterstained with fluorescein-labeled avidin according to our previously published protocol (Botchkarev et al. 1997b ), which specifically highlights mast cells. Cell nuclei were counterstained with DAPI (Mecklenburg et al. 2000 ). Slides incubated with the secondary antibody only served as a negative control. Negative controls showed only background staining similar to background staining in the test slides. Whole-mount embryos served as positive controls, because it is known that these proteins are expressed in the neural crest and bone marrow before skin development.

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 (Botchkarev et al. 1995 ; Paus et al. 1998 ). For each stage of hair follicle morphogenesis, at least 20 hair follicles were studied per mouse (i.e., more than 100 hair follicles from five different mice were studied for each IHC reaction and time point). IR patterns were also recorded qualitatively in schematized recording protocols (Paus et al. 1999 ).

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 (0.5–1-µm resin sections) and by TEM for the presence of cells with ultrastructural features of melanoblasts and melanocytes (Tobin et al. 1998 , Tobin et al. 1999 ). Such melanocytic diagnostic features included premelanosomes (stage 1–3), mature melanosomes (stage 4), absence of keratinocyte-specific tonofilaments, desmosomes, and the presence of clear cytoplasm with extensive Golgi complexes. Melanocytes and melanoblasts were further distinguished from immature undifferentiated keratinocytes by the presence of intercellular junctions in the latter (Tobin and Paus 2001 ).


  Results
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 2A4–2A6). 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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Figures 1-3. c-Kit- and S100-IR and melanocyte ultrastructure in murine back skin hair follicles during hair follicle morphogenesis. Left columns show schematic drawings of c-Kit-IR distribution in the developing hair follicles from stage 0 to stage 8. Arrows point to putative melanoblasts and melanocytes referred to in the corresponding description if not otherwise indicated. c-Kit-IR patterns are demonstrated in columns headed c-Kit and are labeled A plus the number of the respective stage (e.g., A0 for stage 0). Likewise, S100-IR patterns are headed S100 and labeled B HRLM images are headed HRLM and labeled C and TEM images are headed EM and labeled D. White boxes indicate inserts. Dotted lines indicate the basement membrane separating the epithelial follicular and mesenchymal compartment, if not stated otherwise. b, bulge; bv, blood vessel; cs, cytosol; d, dermis; dp, dermal papilla; e, epidermis; hfe, hair follicle epithelium; hm, hair matrix; hs, hair shaft; irs, inner root sheath; mc, mast cell; n, nucleus; nf, nerve fiber; nfb, nerve fiber bundle; ors, outer root sheath; pc, panniculus carnosus; sc, subcutis.

Figure 1. Stage 0 to stage 3 of hair follicle morphogenesis. (A0–D0) Developing epidermis prior to hair follicle development. S100-IR (B0) staining intensity is highest in the basal layer, with single cells showing more prominent staining (arrow; cell has a large cell nucleus and is in close contact with the basement membrane, which is characteristic of melanocytes in the basal layer of the epidermis). Also note the dark heterochromatic nuclei of putative melanoblasts (arrows) by HRLM (C0). One similar looking cell in the suprabasal layer (D0a) contains premelanosomes (D0b) by EM. (A1–D1-2) Stage 1 and stage 1–2 hair follicles. Note the few melanosome-like granules in the mitotic cell with clear cytoplasm (arrow) visible by EM (D1-2) next to a dermal blood vessel. (A2–D2) Stage 2 of hair follicle development. Note the flattened nucleus of a c-Kit-IR cell (arrow) that appears to squeeze a dendrite between the hair follicle epithelial cells in direction of the basement membrane (A2). S100-IR is present in a morphologically similar cell, extending a delicate dendrite into the hair follicle epithelium (B2, arrow). By HRLM a cell in the same location (C2, lower, central arrow) displays the clear cytoplasm typical for melanoblasts, and is caught in mid-mitosis. More melanoblast-like cells with clear cytoplasm and heterochromatic nuclei are located nearby and in the epidermis in touch with the basement membrane (C2, upper two arrows). By EM such cells (D2a, arrow) contain premelanosomes (D2b). (A3–D3b) Stage 3 of hair follicle development. By EM (D3a) a single cell now appears deep on the down-growing hair follicle epithelium suggesting that this cell has originated in the epidermis. This cell contains premelanosomes and melanosomes (D3b, enlargement from D3a indicated by white box). The dotted white line indicates the basement membrane separating the hair follicle epithelium from the surrounding mesenchyme.




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Figure 2. Stage 4–6 of hair follicle morphogenesis. (A4–D4b) Stage 4 of hair follicle development. The dermal papilla is partially invaginated by the hair follicle epithelium and the inner root sheath is beginning to form. Upper dotted lines indicate the inner root sheath. Note the number of c-Kit-IR cells within the hair follicle epithelium (A4). Note the absence of c-Kit-IR cells from just above the dermal papilla, where the inner root sheath is just beginning to form. In the center of the hair follicle epithelium, just above the newly forming inner root sheath, one such cell displays a migratory appearance (A4, arrow). A similar single cell is detected by S100-IR (B4, arrow). Note the absence of S100-IR cells from just above the dermal papilla, where the inner root sheath is just beginning to form. By TEM (D4a) a cell with clear cytoplasm in a comparable localization contains melanosomes (D4b). (A5–D5) Stage 5 of hair follicle development. The pigmented hair shaft is beginning to form. c-Kit-IR cells appear to concentrate in the developing bulge region (compare schematic drawing), and around the dermal papilla (A5, arrow). Note the weak S100-IR in a single cell that appears to have just reached its place of determination contacting the basement membrane above the dermal papilla (B5, arrow). By TEM, cells in similar locations exhibit many dendrites and contain many melanosomes (D5, arrows). (A6–D6) Stage 6 of hair follicle development. The pigmented hair shaft is approaching the developing sebaceous gland. Note the stretched-out morphology of an S100-IR cell that appears to move past the inner root sheath towards the dermal papilla (B6, arrow). By HRLM, two single cells with a clear cytoplasm and heterochromatic nuclei (C6a, arrows) are detected in the isthmus and bulge region (compare schematic drawing).

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 1B0–1B6 and 2B0–2B6) (Egan et al. 1986 ).

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 1–3/4), individual c-Kit-IR cells with few dendrites were observed to increasingly invade the hair follicle epithelium (Fig 1A1–1A4 and 2A1–2A4). 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 (Magerl et al. 2001 ) (Fig 1A3 and 2A4). By contrast, c-Kit-IR cells within the follicular epithelium that had arrived to make contact with the basement membrane of the developing dermal papilla exhibited none or only single dendrites and assumed a more rounded appearance by stage 3 (Fig 1A3). As the developing hair matrix increasingly invaginated the dermal papilla in stage 4, c-Kit-IR cells with two and more dendrites were distributed in the proximal lateral hair bulb on both sides of the dermal papilla. No c-Kit-IR cells were located in the developing precortical region of the hair matrix around which the inner root sheath was beginning to form (Fig 2A4).

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 1B1–1B4 and 2B1–2B4) 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 2–4 (Fig 1C2–1C4, 1D2a–1D4b and 2C2–2C4 and 1D2a–1D4b). 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 1–2 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 (Paus et al. 1999 ). By this time there were increasing numbers of c-Kit-IR cells in several different compartments of the hair follicle epithelium. Some polydendritic c-Kit-IR cells became detectable in the so-called melanogenic zone of the epithelial hair bulb, i.e., above and around the upper third of the dermal papilla (Fig 2A5 and 2A6). However, oligodendritic c-Kit-IR cells continued to be detected in the ORS and bulge region (Fig 2A5 and 2A6), the probable repository of melanocytic and epithelial stem cells (Cotsarelis et al. 1990 ; Tobin and Bystryn 1996 ; Cotsarelis 1997 ; Tobin et al. 1998 ; Taylor et al. 2000 ; Tobin and Paus 2001 ).

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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Figure 3. Stage 7 of hair follicle morphogenesis. (A7–D7) Stage 7 of hair follicle development just before penetration of the epithelium by the developing hair shaft. The hair follicle pigmentary unit is fully established and actively producing pigment for the hair shaft. c-Kit-IR cells are distributed throughout the outer root sheath and in the bulge area. S100-IR is located in Schwann cells accompanying a set of nerve fibers located around the hair follicle epithelium in the space between the sebaceous gland and the insertion of the arrector pili muscle into the bulge region (Botchkarev et al. 1997a ) (B7a enlargement of A7b indicated by white box, and b). It is also located in a subcutaneous nerve fiber bundle (Botchkarev et al. 1997a ) and in the dermal papilla fibroblasts (B7b). However, S100-IR single cells can not be detected within the hair follicle epithelium. By HRLM and EM, melanocytes located around/above the dermal papilla are readily recognizable due to their increasing level of melanogenesis (C7f–D7c). These dendritic cells contain clear expansive cytoplasm with many mitochondria and euchromatic nuclei indicative of increasing metabolic activity and gene transcription respectively (C7f enlargement of C7d indicated by white box, and D7a with enlargement D7b indicated by white box). Melanosome transfer to pericortical/premedullary keratinocytes is now evident (D7b). Melanocytes located elsewhere in the developing hair follicle, e.g., in the ORS, are smaller by contrast, had few dendrites, and exhibited high nuclear to cytoplasmic ratios (C7a, C7b, C7c, C7d, C7e enlargements of A7c and A7f indicated by white boxes, and A7c and A7f). Moreover, the nuclei of these cells are much more heterochromatic than in those located around the dermal papilla, all of which are features of undifferentiated melanocytes. (A8–D8) Stage 8 of hair follicle development. The hair shaft has penetrated the epidermis. c-Kit-IR cells are restricted to the hair bulb (A8). By HRLM and TEM putative melanocytes with clear cytoplasm and heterochromatic nuclei (C8a and C8b) can still be detected in the isthmus and bulge region (arrows) and do contain the occasional melanin granule proving them to be melanocytic (D8a and D8b, arrowheads).



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Figure 4. c-Kit-IR in the bulb of a Stage 8 hair follicle. Dotted lines indicate the basement membrane separating the hair follicle epithelium from the dermal papilla. Continuous lines indicate Auber's line (Tobin et al. 1999 ). Note the negatively stained layer of cells located between the strongly IR cells that are obscured by melanin granules in the upper hair bulb and the equally strongly IR cells displaying a cobblestone-like pattern in the lower bulb. dp, dermal papilla; hm, hair matrix; sc, subcutis.

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) (Botchkarev et al. 1997a ).

HRLM and TEM analysis not only demonstrated that melanocytes were located in the melanogenic zone above and around the upper dermal papilla (Fig 3C7a–3C7f, 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 1–6 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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Figure 5. c-Kit-IR and S100-IR double staining of melanocytic cells within the hair follicle epithelium. This section has been double stained for c-Kit-IR (red fluorescence) and S100-IR (green fluorescence) cells. Dotted line indicates the basement membrane separating the hair follicle epithelium from the dermal papilla of a stage 2 hair follicle. Note the bright yellow fluorescence where the red c-Kit- and green S100-IR overlap (upper arrow) in a dendritic cell in the center of a stage 2 hair follicle. Another cell is double stained (yellow) in the epithelium of a neighboring hair follicle in stage 3–4 (lower arrow) and the S100-IR epidermis appears green. Dotted line indicates the shape of the dermal papilla. dp, dermal papilla; d, dermis; e, epidermis.

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 2A3–2A8 and 5A3–5A8), 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 (Botchkarev et al. 1997b ), revealed many S100-negative mast cells in close contact with S100-IR nerve fiber bundles in the dermis (Fig 6A). A similar double staining of c-Kit with fluorescein-labeled avidin revealed almost complete double labeling of c-Kit-IR dermal cells with the mast cell marker (not shown).



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Figure 6. SCF-IR in murine back skin hair follicle during hair follicle morphogenesis. (A2–A5, A8) SCF-IR patterns detected with the ABC staining technique (for details see Materials and Methods). (B2, B5, B8) SCF-IR patterns detected with the tyramide amplification method (see Materials and Methods). d, dermis; dp, dermal papilla; e, epidermis; hfe, hair follicle epithelium; hm, hair matrix; irs, inner root sheath; sc, subcutis. (A2, B2) All cells of a stage 2 hair follicle are SCF-IR. Note the weaker staining in the dermal papilla. (A3) All cells of a stage 3 hair follicle are SCF-IR. Note the weaker staining in the dermal papilla. (A4) All cells of a stage 4 hair follicle are SCF-IR. Note the weaker staining in the inner root sheath and the dermal papilla and the positive staining in dermal fibroblasts. (A5, B5) All cells of a stage 5 hair follicle are SCF-IR. Note the strong staining in the outer root sheath and hair matrix and the weaker staining in the inner root sheath and dermal papilla. (A8, B8) All cells of a stage 8 hair follicle are SCF-IR. Note the strong staining in the outer root sheath and hair matrix and the weaker staining in the inner root sheath and dermal papilla.

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).


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Ito et al. 1993 ). (c) S100-IR cells gradually disappear from the hair follicle epithelium after the onset of bulbar pigment production. (d) c-Kit-IR cells populating the epithelial hair bulb display a progressively dendritic phenotype and increase in numbers from the earliest stages of follicle development. (e) By HRLM and TEM, cells in c-Kit-IR hair follicle locations above Auber's line lack epithelial cell specialization, including tonofilaments and desmosomes, but increasingly display melanocytic features such as premelanosomes and melanosomes.

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 (Paus et al. 1998 ), the latter of which can be excluded because they contain cell-specific racket-shaped granules and are rarely found in the hair follicle below the sebaceous gland (Paus et al. 1998 ), even in human hair follicles (Moresi and Horn 1997 ; Christoph et al. 2000 ).

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 Paus et al. 1996 ).

Most interestingly, the S100 antibody employed here to detect melanocytic cells of neural crest origin (Ito et al. 1993 ) within the hair follicle epithelium also detected differentiating epithelial cells of the epidermis, dermal papilla fibroblasts of fully developed hair follicles, and Schwann cells (data not shown; and Peters et al. in press ). This again demonstrates the wide variety of S100 proteins and their multiple roles in neural crest-derived cell development (Lesniak et al. 2000 ), epithelial differentiation (Mischke et al. 1996 ), and fibroblast development (Klingelhofer et al. 1997 ; Lesniak et al. 2000 ). Therefore, it is necessary to interpret carefully the individual S-100 staining patterns in the context of their localization. S100-IR in the epidermis, where Ca-binding S100 proteins are present in abundance (Shrestha et al. 1998 ), may have masked S100-IR in neural crest-derived cell populations within this compartment. However, the hair follicle epithelium was S100-negative except for single scattered cells. These latter cells were identified as melanoblasts with an appearance suggestive of actively migrating cells by their IR to c-Kit, their morphological appearance, and their localization in hair follicle compartments containing ultrastructurally identifiable melanoblasts. Moreover, Schwann cells, fibroblasts, and Langerhans cells were absent from the hair follicle locations containing S100/c-Kit-IR cells. Thus, in the developing hair follicle, S100 is a valid marker for neural crest-derived melanocytic cells before their differentiation.

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 (Kunisada et al. 1998a ). Melanoblasts are detectable in mice that are deficient in the membrane-bound isoform of SCF. These cells fail to disperse into the developing skin, resulting in white coat color (Brannan et al. 1991 ; Wehrle-Haller and Weston 1995 ).

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 (Wehrle-Haller and Weston 1999 ). Reduced SCF levels in the epidermis (Besmer et al. 1993 ) or increased competition for SCF by ectopic c-Kit expression in the somites (Duttlinger et al. 1993 ) also results in a lack of pigmented hair shafts in mutant mice, despite normal development and distribution of melanoblasts before hair follicle morphogenesis. By contrast, overexpression of the membrane-bound SCF isoform under the K14 promotor (Kunisada et al. 1998a ), or SCF release from beads implanted into organ-cultured skin, results in follicular and epidermal hypermelanosis (Jordan and Jackson 2000 ). Overexpression of the soluble and membrane bound isoforms under the K14 promotor produces additional mastocytosis (Kunisada et al. 1998a ).

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 (Imokawa et al. 1996 ; Ono et al. 1998 ) or specific adhesion molecules such as ß1 integrin (Scott et al. 1994 ; Beauvais-Jouneau et al. 1999 ).

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 (Nishikawa et al. 1991 ; Yoshida et al. 1996 ). When such antibodies are injected later, only patches of white hair develop between the pigmented hair shafts. Melanocytes were only detected in at least stage 4 hair follicles in contact with the epidermal or follicular basement membrane shortly after treatment (Yoshida et al. 1996 ).

These findings concur with our understanding of the development of coat color in successive waves of hair follicle generations (Nishikawa et al. 1991 ; Yoshida et al. 1996 ). They also demonstrate the reliance of migrating melanocytes on c-Kit/SCF signaling, independent of basement membrane contact. Moreover, these features appear to be repeated during adult hair follicle cycling, as demonstrated by the observation that the administration of c-Kit-neutralizing antibodies to adult depilated mice interferes with the activation of melanocytes during anagen (Nishikawa et al. 1991 ; Botchkareva et al. 2001 ).

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 hematology–oncology patients. Few side effects were experienced other than a flare and wheal reaction and focal hyperpigmentation at the site of subcutaneous injection (Grichnik et al. 1995 ; Costa et al. 1996 ; Shpall 1999 ). This interesting observation suggests that local injection of SCF may well be exploitable in the treatment of poliosis as seen, e.g., in association with vitiligo and alopecia areata (Tobin et al. 1990 ), provided that the hair follicle still contains a sufficient pool of melanocytic cells capable of responding to SCF stimulation (Nordlund et al. 1998 ).


  Acknowledgments

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.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Beauvais–Jouneau A, Pla P, Bernex F, Dufour S, Salamero J, Fassler R, Panthier JJ, Thiery JP, Larue L (1999) A novel model to study the dorsolateral migration of melanoblasts. Mech Dev 89:3-14[Medline]

Besmer P, Manova K, Duttlinger R, Huang EJ, Packer A, Gyssler C, Bachvarova RF (1993) The kit-ligand (steel factor) and its receptor c-kit/W: pleiotropic roles in gametogenesis and melanogenesis. Development Suppl 125–137

Botchkareva NV, Khlgatian M, Longley BJ, Botchkarev VA, Gilchrest BA (2001) SCF/c-kit signaling is required for cyclic regeneration of the hair pigmentation unit. FASEB J 15:645-658[Abstract/Free Full Text]

Botchkarev VA, Eichmüller S, Johansson O, Paus R (1997a) Hair cycle-dependent plasticity of skin and hair follicle innervation in normal murine skin. J Comp Neurol 386:379-395[Medline]

Botchkarev VA, Eichmüller S, Peters EM, Pietsch P, Johansson O, Maurer M, Paus R (1997b) A simple immunofluorescence technique for simultaneous visualization of mast cells and nerve fibers reveals selectivity and hair cycle-dependent changes in mast cell-nerve fiber contacts in murine skin. Arch Dermatol Res 289:292-302[Medline]

Botchkarev VA, Paus R, Czarnetzki BM, Kupriyanov VS, Gordon DS, Johansson O (1995) Hair cycle-dependent changes in mast cell histochemistry in murine skin. Arch Dermatol Res 287:683-686[Medline]

Brannan CI, Lyman SD, Williams DE, Eisenman J, Anderson DM, Cosman D, Bedell MA, Jenkins NA, Copeland NG (1991) Steel-Dickie mutation encodes a c-kit ligand lacking transmembrane and cytoplasmic domains. Proc Natl Acad Sci USA 88:4671-4674[Abstract]

Christoph T, Müller–Röver S, Audring H, Tobin DJ, Hermes B, Cotsarelis G, Ruckert R, Paus R (2000) The human hair follicle immune system: cellular composition and immune privilege. Br J Dermatol 142:862-873[Medline]

Costa JJ, Demetri GD, Harrist TJ, Dvorak AM, Hayes DF, Merica EA, Menchaca DM, Gringeri AJ, Schwartz LB, Galli SJ (1996) Recombinant human stem cell factor (kit ligand) promotes human mast cell and melanocyte hyperplasia and functional activation in vivo. J Exp Med 183:2681-2686[Abstract]

Cotsarelis G (1997) The hair follicle: dying for attention. Am J Pathol 151:1505-1509[Medline]

Cotsarelis G, Sun TT, Lavker RM (1990) Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 61:1329-1337[Medline]

Duttlinger R, Manova K, Chu TY, Gyssler C, Zelenetz AD, Bachvarova RF, Besmer P (1993) W-sash affects positive and negative elements controlling c-kit expression: ectopic c-kit expression at sites of kit-ligand expression affects melanogenesis. Development 118:705-717[Abstract/Free Full Text]

Egan MJ, Crocker J, Newman J, Collard M (1986) Immunohistochemical localization of S100 protein in skin tumors. Arch Pathol Lab Med 110:765-767[Medline]

Eichmüller S, Stevenson PA, Paus R (1996) A new method for double immunolabelling with primary antibodies from identical species. J Immunol Methods 190:255-265[Medline]

Eichmüller S, van der Veen C, Moll I, Hermes B, Hofmann U, Müller–Röver S, Paus R (1998) Clusters of perifollicular macrophages in normal murine skin: physiological degeneration of selected hair follicles by programmed organ deletion. J Histochem Cytochem 46:361-370[Abstract/Free Full Text]

Grabbe J, Welker P, Dippel E, Czarnetzki BM (1994) Stem cell factor, a novel cutaneous growth factor for mast cells and melanocytes. Arch Dermatol Res 287:78-84[Medline]

Grichnik JM, Ali WN, Burch JA, Byers JD, Garcia CA, Clark RE, Shea CR (1996) KIT expression reveals a population of precursor melanocytes in human skin. J Invest Dermatol 106:967-971[Abstract]

Grichnik JM, Crawford J, Jimenez F, Kurtzberg J, Buchanan M, Blackwell S, Clark RE, Hitchcock MG (1995) Human recombinant stem-cell factor induces melanocytic hyperplasia in susceptible patients. J Am Acad Dermatol 33:577-583[Medline]

Hoffman U, Tokura Y, Nishijima T, Takigawa M, Paus R (1996) Hair cycle-dependent changes in skin immune functions: anagen-associated depression of sensitization for contact hypersensitivity in mice. J Invest Dermatol 106:598-604[Abstract]

Imokawa G, Yada Y, Kimura M (1996) Signalling mechanisms of endothelin-induced mitogenesis and melanogenesis in human melanocytes. Biochem J 314:305-312[Medline]

Ito M, Kawa Y, Ono H, Okura M, Baba T, Kubota Y, Nishikawa SI, Mizoguchi M (1999) Removal of stem cell factor or addition of monoclonal anti-c-KIT antibody induces apoptosis in murine melanocyte precursors. J Invest Dermatol 112:796-801[Abstract/Free Full Text]

Ito K, Morita T, Sieber–Blum M (1993) In vitro clonal analysis of mouse neural crest development. Dev Biol 157:517-525[Medline]

Jackson IJ (1994) Molecular and developmental genetics of mouse coat color. Annu Rev Genet 28:189-217[Medline]

Jordan SA, Jackson IJ (2000) MGF (KIT ligand) is a chemokinetic factor for melanoblast migration into hair follicles. Dev Biol 225:424-436[Medline]

Klingelhofer J, Ambartsumian NS, Lukanidin EM (1997) Expression of the metastasis-associated mts1 gene during mouse development. Dev Dyn 210:87-95[Medline]

Kunisada T, Lu SZ, Yoshida H, Nishikawa S, Nishikawa S, Mizoguchi M, Hayashi S, Tyrrell L, Williams DA, Wang X, Longley BJ (1998a) Murine cutaneous mastocytosis and epidermal melanocytosis induced by keratinocyte expression of transgenic stem cell factor. J Exp Med 187:1565-1573[Abstract/Free Full Text]

Kunisada T, Yoshida H, Yamazaki H, Miyamoto A, Hemmi H, Nishimura E, Shultz LD, Nishikawa S, Hayashi S (1998b) Transgene expression of steel factor in the basal layer of epidermis promotes survival, proliferation, differentiation and migration of melanocyte precursors. Development 125:2915-2923[Abstract/Free Full Text]

Lahav R, Lecoin L, Ziller C, Nataf V, Carnahan JF, Martin FH, Le Douarin NM (1994) Effect of the Steel gene product on melanogenesis in avian neural crest cell cultures. Differentiation 58:133-139[Medline]

Lesniak W, Swart GW, Bloemers HP, Kuznicki J (2000) Regulation of cell specific expression of calcyclin (S100A6) in nerve cells and other tissues. Acta Neurobiol Exp 60:569-575[Medline]

Luo D, Chen H, Searles G, Jimbow K (1995) Coordinated mRNA expression of c-Kit with tyrosinase and TRP-1 in melanin pigmentation of normal and malignant human melanocytes and transient activation of tyrosinase by Kit/SCF-R. Melanoma Res 5:303-309[Medline]

Magerl M, Tobin DJ, Müller-Röver S, Hagen E, Lindner G, McKay IA, Paus R (2001) Patterns of proliferation and apoptosis during murine hair follicle morphogenesis. J Invest Dermatol 116:947-955[Abstract/Free Full Text]

Maurer M, Fischer E, Handjiski B, von Stebut E, Algermissen B, Bavandi A, Paus R (1997) Activated skin mast cells are involved in murine hair follicle regression (catagen). Lab Invest 77:319-332[Medline]

Maurer M, Paus R, Czarnetzki BM (1995) Mast cells as modulators of hair follicle cycling. Exp Dermatol 4:266-271[Medline]

Mecklenburg L, Tobin DJ, Müller–Röver S, Handjiski B, Wendt G, Peters EM, Pohl S, Moll I, Paus R (2000) Active hair growth (anagen) is associated with angiogenesis. J Invest Dermatol 114:909-916[Abstract/Free Full Text]

Mischke D, Korge BP, Marenholz I, Volz A, Ziegler A (1996) Genes encoding structural proteins of epidermal cornification and S100 calcium-binding proteins form a gene complex "epidermal differentiation complex" on human chromosome 1q21. J Invest Dermatol 106:989-992[Abstract]

Moresi JM, Horn TD (1997) Distribution of Langerhans cells in human hair follicle. J Cutan Pathol 24:636-640[Medline]

Motro B, van der Kooy D, Rossant J, Reith A, Bernstein A (1991) Contiguous patterns of c-kit and steel expression: analysis of mutations at the W and Sl loci. Development 113:1207-1221[Abstract]

Müller–Röver S, Handjiski B, van der Veen C, Eichmüller S, Foitzik K, McKay IA, Stenn KS, Paus R (2001) A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. J Invest Dermatol 117:3-15[Abstract/Free Full Text]

Nishikawa S, Kusakabe M, Yoshinaga K, Ogawa M, Hayashi S, Kunisada T, Era T, Sakakura T, Nishikawa S (1991) In utero manipulation of coat color formation by a monoclonal anti-c-kit antibody: two distinct waves of c-kit-dependency during melanocyte development. EMBO J 10:2111-2118[Abstract]

Nordlund JJ, Boissy RE, Hearing VJ, King RA, Ortonne J-P (1998) The Pigmentary System. New York, Oxford, Oxford University Press

Ono H, Kawa Y, Asano M, Ito M, Takano A, Kubota Y, Matsumoto J, Mizoguchi M (1998) Development of melanocyte progenitors in murine Steel mutant neural crest explants cultured with stem cell factor, endothelin-3, or TPA. Pigment Cell Res 11:291-298[Medline]

Paus R, Müller–Röver S, van Der Veen C, Maurer M, Eichmüller S, Ling G, Hofmann U, Foitzik K, Mecklenburg L, Handjiski B (1999) A comprehensive guide for the recognition and classification of distinct stages of hair follicle morphogenesis. J Invest Dermatol 113:523-532[Abstract/Free Full Text]

Paus R, Peters EM, Eichmüller S, Botchkarev VA (1997) Neural mechanisms of hair growth control. J Invest Dermatol Symp Proc 2:61-68

Paus R, van der Veen C, Eichmüller S, Kopp T, Hagen E, Müller–Röver S, Hofmann U (1998) Generation and cyclic remodeling of the hair follicle immune system in mice. J Invest Dermatol 111:7-18[Abstract]

Paus R, Welker P, Jensen K, Handjiski B, Eichmüller S, Botchkarev VA, Maurer M, Scott GA (1996) Intraepithelial C-kit expression during murine hair follicle development and cycling: a role for stem cell factor in epithelial biology. J Invest Dermatol 106:834

Peters EMJ, Botchkarev VA, Rice FL, Müller–Röver S, Moll I, Paus R (in press) Development of back skin and hair follicle innervation: a comprehensive study of neuronal related structural proteins, enzymes, and peptides during murine hair follicle morphogenesis. J Comp Neurol

Schindler KS, Roth KA (1996) Double immunofluorescent staining using two unconjugated primary antisera raised in the same species. J Histochem Cytochem 44:1331-1335[Abstract/Free Full Text]

Scott G, Ewing J, Ryan D, Abboud C (1994) Stem cell factor regulates human melanocyte-matrix interactions. Pigment Cell Res 7:44-51[Medline]

Shpall EJ (1999) The utilization of cytokines in stem cell mobilization strategies. Bone Marrow Transpl 23:S13-19[Medline]

Shrestha P, Muramatsu Y, Kudeken W, Mori M, Takai Y, Ilg EC, Schafer BW, Heizmann CW (1998) Localization of Ca(2+)-binding S100 proteins in epithelial tumours of the skin. Virchows Arch 432:53-59[Medline]

Slominski A, Paus R (1993) Melanogenesis is coupled to murine anagen: toward new concepts for the role of melanocytes and the regulation of melanogenesis in hair growth. J Invest Dermatol 101:90S-97S[Abstract]

Slominski A, Paus R, Schadendorf D (1993) Melanocytes as "sensory" and regulatory cells in the epidermis. J Theor Biol 164:103-120[Medline]

Taylor G, Lehrer MS, Jensen PJ, Sun TT, Lavker RM (2000) Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell 102:451-461[Medline]

Tobin DJ, Bystryn JC (1996) Different populations of melanocytes are present in hair follicles and epidermis. Pigment Cell Res 9:304-310[Medline]

Tobin DJ, Fenton DA, Kendall MD (1990) Ultrastructural observations on the hair bulb melanocytes and melanosomes in acute alopecia areata. J Invest Dermatol 94:803-807[Abstract]

Tobin DJ, Hagen E, Botchkarev VA, Paus R (1998) Do hair bulb melanocytes undergo apoptosis during hair follicle regression (catagen)? J Invest Dermatol 111:941-947[Abstract]

Tobin DJ, Paus R (2001) Graying: gerontobiology of the hair follicle pigmentary unit. Exp Gerontol 36:29-54[Medline]

Tobin DJ, Slominski A, Botchkarev V, Paus R (1999) The fate of hair follicle melanocytes during the hair growth cycle. J Invest Dermatol Symp Proc 4:323-332

van Gijlswijk RP, Zijlmans HJ, Wiegant J, Bobrow MN, Erickson TJ, Adler KE, Tanke HJ, Raap AK (1997) Fluorochrome-labeled tyramides: use in immunocytochemistry and fluorescence in situ hybridization. J Histochem Cytochem 45:375-382[Abstract/Free Full Text]

Wehrle–Haller B, Weston JA (1995) Soluble and cell-bound forms of steel factor activity play distinct roles in melanocyte precursor dispersal and survival on the lateral neural crest migration pathway. Development 121:731-742[Abstract/Free Full Text]

Wehrle–Haller B, Weston JA (1999) Altered cell-surface targeting of stem cell factor causes loss of melanocyte precursors in Steel 17H mutant mice. Dev Biol 210:71-86[Medline]

Yoshida H, Kunisada T, Kusakabe M, Nishikawa S, Nishikawa SI (1996) Distinct stages of melanocyte differentiation revealed by analysis of nonuniform pigmentation patterns. Development 122:1207-1214[Abstract/Free Full Text]