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Fate of Melanocytes During Development of the Hair Follicle Pigmentary Unit
ORIGINAL ARTICLE
Fate of Melanocytes During Development of the Hair Follicle Pigmentary Unit Natalia V. Botchkareva,1 Vladimir A. Botchkarev, and Barbara A. Gilchrest Department of Dermatology, Boston University School of Medicine, Boston, MA, USA
During hair follicle morphogenesis, melanocyte precursors migrate into developing hair follicles and give rise to di¡erentiated melanocytes that actively produce and transport pigment into the keratinocytes that form the hair shaft; however, patterns of melanocyte proliferation and di¡erentiation during formation of the hair pigmentation unit remain to be elucidated. Using multicolor confocal microscopy and double immuno£uorescence of melanogenic proteins (tyrosinase-related proteins 1 and 2, tyrosinase) and the proliferative marker Ki67, we have studied melanocyte development in C57BL/6 mouse embryonic hair follicles. Proliferating melanocyte precursors (tyrosinase-related protein-2/ Ki67 þ cells) are seen in the hair follicles at stages 1^2 of morphogenesis, as follicular invagination begins. In stage 3^4 hair follicles, the majority of intrafollicular melanocytes remain tyrosinase-related protein-2 þ and Ki67 þ, whereas some located adjacent to the forming dermal papilla begin to express tyrosinase-related protein-1, an early marker of di¡erentiation. Melanin gran-
ules appear in stage 5 hair follicles coincident with tyrosinase expression in nonproliferating tyrosinase-related protein-2 þ /tyrosinase-related protein-1 þ melanocytes. Stage 6^8 hair follicles, those actively producing hair, show nonproliferating tyrosinase-related protein2 þ melanocytes in the bulge area, tyrosinase-related protein-2 þ /tyrosinase-related protein-1 þ melanocytes in the outer root sheath, and tyrosinase-related protein-2 þ /tyrosinase-related protein-1 þ /tyrosinase þ melanocytes above the dermal papilla. These data suggest that melanocyte precursor cells proliferate extensively at the onset of follicle development. Progeny of these cells migrate down the developing follicle, differentiating further until reaching the area immediately above the dermal papilla, where fully di¡erentiated nonproliferative melanin-producing melanocytes persist, contributing pigment to the growing hair shaft. Key words: morphogenesis/skin neuralcrest/melanin. JID Symposium Proceedings 8:76 ^79, 2003
H
Melanogenesis is controlled by several key enzymes that are uniquely expressed in melanocytes (reviewed in Hearing, 1999). Tyrosinase catalyzes the rate-limiting initial events of melanogenesis, and mutations in tyrosinase gene lead to loss of pigment (Beermann et al, 1990). Tyrosinase-related proteins (TRP) 1 and TRP2 share 40^45% amino acid identity with tyrosinase and are also critically important for melanogenesis, functioning as downstream enzymes in the melanin biosynthetic pathway (Winder et al, 1994); and mutations in these genes also a¡ect pigmentation in mice. In postnatal murine anagen HF, melanocytes are divided into three distinct subpopulations. The ¢rst is located in the HF bulge, expresses only TRP2, does not proliferate, and presumptively represents melanocyte stem cells. The second is located in the HF outer root sheath, expresses TRP2 and relatively weak TRP1, displays proliferative activity, and represents di¡erentiating melanocytes. The third is located in the hair matrix above the DP, expresses TRP2, TRP1, and tyrosinase, does not proliferate, and actively produces melanin (Tobin et al, 1999; Botchkareva et al, 2001). The dynamics of the formation of HF pigmentary units in developing HF is only poorly understood, however. As shown by immunohistochemical and transgenic studies, melanocytes in the developing HF express TRP2 and can be identi¢ed as early as stage 2 HF (Yoshida et al, 1996; Jordan and Jackson, 2000). It is unknown whether these melanocytes proliferate during transition to more di¡erentiated forms, and whether melanogenic
air follicle (HF) development is driven by inductive interactions between epidermal keratinocytes committed to hair-speci¢c di¡erentiation, and a specialized population of dermal ¢broblasts, which form a condensation beneath the hair placode and subsequently develop into follicular dermal papilla (DP) (reviewed in Philpott and Paus, 1998, Paus, 1998). During HF morphogenesis, neural crest-derived melanocytes migrate into the HF, produce and transport melanin to the keratinocytes of the precortical zone that di¡erentiate to form the pigmented hair shaft (reviewed in Starico, 1960, Hirobe, 1995, Lecoin et al, 1999). In developing HF, melanocyte migration and melanogenic activity are controlled by stem cell factor and its receptor c-kit. Loss of function for these proteins either in corresponding mouse mutants or after treatment with c-kit neutralizing antibody results in the formation of nonpigmented hairs (Chabot et al, 1988, Zsebo et al, 1990, Nishikawa et al, 1991, Yoshida et al, 1996, Jordan and Jackson, 2000).
Manuscript accepted for publication February 1, 2003 Reprint requests to: Barbara A. Gilchrest, Department of Dermatology, Boston University School of Medicine, 609 Albany Street, Boston, MA 02118, U.S.A. Email: [email protected] 1 Present address: The Gillette Company, Needham, MA, U.S.A. Abbreviations: HF, hair follicle; DP, dermal papilla; TRP, tyrosinaserelated protein.
0022-202X/03/$15.00 . Copyright r 2003 by The Society for Investigative Dermatology, Inc. 76
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Figure 1. Expression patterns for melanogenic proteins during hair follicle morphogenesis. Cryosections of back skin of E16.5^18.5 or P0.5^6.5 C57BL/6 mice were immunostained with anti-sera against proliferative marker Ki- 67 (red £uorescence) and one of the melanogenic proteins (TRP1,TRP2, or tyrosinase; green £uorescence). Slides were counterstained with TO-PRO-3 (blue £uorescence). Left column of ¢gures shows schematic drawings of distinct stages of hair follicle development (Paus et al, 1999). (A) Colocalization of Ki- 67 and TRP2 (arrow) in undi¡erentiated melanocytes of stage 1 HF. Dermal^epidermal border is indicated by dotted line. (B) Colocalization of Ki- 67 and TRP2 (arrows) in undi¡erentiated melanocytes of stage 3 HF. (C) Lack of Ki- 67 in TRP1 þ di¡erentiating melanocytes (arrow) of stage 3 HF. (D) Colocalization of Ki- 67 and TRP2 in melanocytes located in central portion of stage 4 HF (arrow). Lack of Ki- 67 in TRP2 þ melanocytes located close to the DP (arrowhead) of stage 5 HF. (E) Lack of Ki- 67 in TRP1 þ melanocytes located close to the DP (arrowhead) of stage 5 HF. (F) Hair bulb melanocytes of fully developed stage 8 HF express tyrosinase and are Ki- 67 (arrows).
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JID SYMPOSIUM PROCEEDINGS
activity of HF melanocytes detected at more advanced stages of HF morphogenesis (Hirobe, 1995, Paus et al, 1999) is associated with changes in expression of other melanogenic proteins (TRP1, tyrosinase). We have now systematically analyzed the expression of TRP1, TRP2, and tyrosinase in proliferating, di¡erentiating, and melanogenically active melanocytes during HF development in normal C57BL/6 mice. This study provides evidence that HF development and formation of the HF pigmentary unit are highly synchronized, and every distinct stage of HF development is associated with its own patterns of melanocyte proliferation, di¡erentiation, and melanogenic activity. MATERIALS AND METHODS Animals and tissue collection C57BL/6 mice were purchased from Charles River (Cambridge, MA) and housed in community cages at the animal facility of the Boston University School of Medicine. For the analysis of HF morphogenesis, embryonic and neonatal skin was harvested at various time points of estimated gestational age [embryonic days (E) 14.5, E16.5, and E18.5], or postnatal age [postnatal days (P) 0, P1, P2, P3, P6, P10 after birth]. For immunohistochemical analysis, the back skin was harvested parallel to the vertebral line embedded rapidly and snap-frozen in liquid nitrogen, using a special technique for obtaining longitudinal cryosections through the HF from one de¢ned site (Paus et al, 1999). Immunohistochemistry and multicolor confocal microscopy For immunohistochemical analyses, acetone-¢xed cryostat sections (8 mm) of adolescent C57BL/6 mouse back skin were used. To study the expression of tyrosinase, TRP1, or TRP2, we performed an immuno£uorescence protocol using the corresponding primary rabbit anti-sera (gift of Dr V. Hearing) and secondary tetramethylrhodamine isothiocyanate-conjugated goat-anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA), as described previously (Botchkareva et al, 2001). For double immunovisualization of melanogenic proteins and the proliferative marker Ki- 67, cryosections were ¢rst incubated with rat monoclonal antibody against murine Ki- 67 (Dianova, Hamburg, Germany), following by incubation with tetramethylrhodamine isothiocyanate-labeled goat anti-rat IgG (Jackson ImmunoResearch). After subsequent washing in Tris-bu¡ered saline, sections were incubated with primary rabbit anti-serum against one of the melanogenic proteins (TRP1, TRP2, or tyrosinase) followed by incubation with £uorescein isothiocyanate-conjugated secondary goatanti-rabbit rat IgG (Jackson ImmunoResearch). Finally, sections were counterstained by TO-PRO3 -iodide (Molecular Probes Inc., Eugene, OR) for visualization of cell nuclei. All slides were analyzed using a Zeiss confocal microscope, and were photo-documented using a digital image analysis system (Pixera, San-Diego, CA).
RESULTS AND DISCUSSION
To de¢ne distribution and proliferative activity of melanocytes during formation of the hair pigmentary unit, we performed double immuno-visualization of melanogenic proteins (TRP1, TRP2, tyrosinase) and the proliferative marker Ki- 67 in cryosections of C57BL/6 murine embryonic (E14.5^18.5) and neonatal (P0.5^10) skin. We found that TRP2 þ and Ki- 67 þ melanocytes were already seen in stage 1 HF as located among keratinocytes of the hair placode (Fig 1A); however, those melanocytes were TRP1^ and tyrosinase^ (not shown), consistent with minimal di¡erentiation. In stage 3 HF, many TRP2 þ melanocytes were visible in the elongating HF and showed Ki- 67 expression (Fig 1B), consistent with migration of these cells down the developing follicle. Single TRP2 þ and Ki- 67^ cells were also seen in stage 3 HF (not shown). TRP1 expression was ¢rst detected in single cells of stage 3 HF, most of them were Ki- 67^ (Fig 1C), consistent with progressive melanocyte di¡erentiation. In stage 5 HF, numerous TRP2 þ and TRP1 þ melanocytes were located immediately above the DP and did not show Ki- 67 expression (Fig 1D,E). Single melanocytes located here were also tyrosinase þ and Ki- 67^ (not shown). During HF progression to the latest stages of development (stages 6^8), tyrosinase expression in the melanocytes located above DP was strongly enhanced
Figure 2. Fate of melanocytes during morphogenesis of hair follicle pigmentary unit. TRP2 þ melanocytes precursor cells (melanocytes stem cells) proliferate extensively at the onset of follicle development. Di¡erentiation of their progeny is associated with inhibition of proliferation and the onset of TRP1 expression. Fully di¡erentiated nonproliferative melanin-producing melanocytes that contribute pigment to the growing hair shaft express all three melanogenic proteins.
(Fig 1F). These melanocytes were also TRP2 þ and TRP1 þ and Ki- 67^, consistent with full di¡erentiation; however, no tyrosinase expression was seen in single TRP2 þ and TRP1 þ melanocytes located in the HF outer root sheath (not shown). Our data and reports published previously (Yoshida et al, 1996, Jordan and Jackson, 2000) suggest that the genetic program of formation of the hair pigmentary unit is organized as a multistep transition of undi¡erentiated melanocytes progenitors to fully di¡erentiated cells actively producing and transporting melanin to hair shaft keratinocytes (Fig 2). As we showed here (Fig 1), this process is highly co-ordinated with distinct morphogenic steps of HF development. Most likely, HF melanocytes arise from the TRP2 þ progenitors that are located in embryonic epidermis (Nishikawa et al, 1991, Yoshida et al, 1996; Jordan and Jackson, 2000). These presumptive stem cells then migrate into developing hair placode (stages 1^2 of HF morphogenesis). At this stage they express TRP2, but do not express the di¡erentiation markers TRP1 and tyrosinase, and proliferate (Figs 1 and 2). Transition of TRP2 þ melanocytes to a more di¡erentiated status is associated with the onset of TRP1 expression and with reduction of their proliferation, as it is visible at stage 3 HF (Fig 1). The onset of melanogenic activity, which is detected in those melanocytes that are closely adjacent to the DP of stage 5 HF (Hirobe, 1995, Paus et al, 1999), is accompanied with the appearance of tyrosinase expression (Figs 1 and 2). Interestingly, tyrosinase expression and melanogenic activity is only detected in the subpopulation of HF melanocytes closest to the DP. This suggest that DP-secreted growth factors, such as stem cell factor, hepatocyte growth factor, and Wnt, are involved in the transition of HF melanocytes to fully di¡erentiated forms that actively produce melanin (Lindner et al, 1999, Botchkareva et al, 2001; Reddy et al, 2001). Therefore, this study provides important basic information about spatiotemporal organization of the HF pigmentary unit during HF development. These data also suggest that formation of the HF pigmentary unit is tightly coupled to distinct morphogenetic steps of HF development and that genetic programs of di¡erentiation of follicular keratinocytes, melanocytes, and DP ¢broblasts are highly synchronized. Molecular mechanisms that are involved in interactions between cells of HF epithelium and mesenchyme with follicular melanocytes during HF development, however, remain to be elucidated.
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REFERENCES Beermann F, Ruppert S, Hummler E, Bosch FX, Mˇller G, Rˇther U, Schˇtz G: Rescue of the albino phenotype by introduction of a functional tyrosinase gene into mice. EMBO J 9:2819^2826, 1990 Botchkareva NV, Khlgatian M, Longley BJ, Botchkarev VA, Gilchrest BA: SCF/c-kit signaling is required for cyclic regeneration of hair pigmentation unit. FASEB J 15:645^658, 2001 Chabot B, Stephenson DA, Chapman VM, Besmer P, Bernstein A: The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus. Nature 335:88^89, 1988 Hearing VJ: Biochemical control of melanogenesis and melanosomal organization. J Invest Dermatol Symp Proc 4:24^28, 1999 Hirobe T: Structure and function of melanocytes: microscopic morphology and cell biology of mouse melanocytes in the epidermis and hair follicle. Histol Histopathol 10:223^237, 1995 Jordan SA, Jackson IJ: MGF (Kit ligand) is a chemokinetic factor for melanoblast migration into hair follicles. Dev Biol 225:424^436, 2000 Lecoin L, Dupin E, LeDouarin N: Development of melanocytes from neural crest progenitors. In: Chuong C-M (ed.). Molecular Basis of Epithelial Appendage Morphogenesis. Austin, TX: R.G. Landes Co., 1999:pp 131^154 Lindner G, Menrad A, Gehrardi E, et al: Involvement of hepatocyte growth factor/ scatter factor and Met receptor signaling in hair follicle morphogenesis and cycling. FASEB J 14:319^332, 1999 Nishikawa S, Kusakabe M, Yoshinaga K, et al: In utero manipulation of coat color formation by a monoclonal anti-c-kit antibody: Two distinct waves of c-kitdependency during melanocyte development. EMBO J 10:2111^2118, 1991
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Paus R: Principles of hair cycle control. J Dermatol Sci 25:793^802, 1998 Paus R, Muller-Rover S, Van Der Veen C, et al: A comprehensive guide for the recognition and classi¢cation of distinct stages of hair follicle morphogenesis. J Invest Dermatol 113:523^532, 1999 Philpott MP, Paus R: Principles of hair follicle morphogenesis. In: Chuong C-M (ed.). Molecular Basis of Epithelial Appendage Morphogenesis. Austin, TX: R.G. Landes Co., 1999:pp 75^103 Reddy S, Andl T, Bagasra A, Lu MM, Epstein DJ, Morrisey EE: SEM Characterization of Wnt gene expression in developing and postnatal hair follicles and identi¢cation of Wnt5a as a target of Sonic hedgehog in hair follicle morphogenesis. Mech Dev 107:69^82, 2001 Starico R: The melanocytes and the hair follicle. J Invest Dermatol 35:185^194, 1960 Tobin DJ, Slominski A, Botchkarev V, Paus R: The fate of hair follicle melanocytes during the hair growth cycle. J Invest Dermatol Symp Proc 4:323^332, 1999 Winder A, Kobayashi T, Tsukamoto K, Urabe K, Aroca P, Kameyama K, Hearing VJ: The tyrosinase gene familyinteractions of melanogenic proteins to regulate melanogensis. Cell Mol Biol Res 40:613^626, 1994 Yoshida H, Kunisada T, Kusakabe M, Nishikawa S, Nishikawa SI: Distinct stages of melanocyte di¡erentiation revealed by analysis of nonuniform pigmentation patterns. Development 122:1207^1214, 1996 Zsebo KM, Williams DA, Geissler EN, et al: Stem cell factor is encoded at the S1 locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor. Cell 63:213^224, 1990
Fate of Melanocytes During Development of the Hair Follicle Pigmentary Unit Natalia V. Botchkareva,1 Vladimir A. Botchkarev, and Barbara A. Gilchrest Department of Dermatology, Boston University School of Medicine, Boston, MA, USA
During hair follicle morphogenesis, melanocyte precursors migrate into developing hair follicles and give rise to di¡erentiated melanocytes that actively produce and transport pigment into the keratinocytes that form the hair shaft; however, patterns of melanocyte proliferation and di¡erentiation during formation of the hair pigmentation unit remain to be elucidated. Using multicolor confocal microscopy and double immuno£uorescence of melanogenic proteins (tyrosinase-related proteins 1 and 2, tyrosinase) and the proliferative marker Ki67, we have studied melanocyte development in C57BL/6 mouse embryonic hair follicles. Proliferating melanocyte precursors (tyrosinase-related protein-2/ Ki67 þ cells) are seen in the hair follicles at stages 1^2 of morphogenesis, as follicular invagination begins. In stage 3^4 hair follicles, the majority of intrafollicular melanocytes remain tyrosinase-related protein-2 þ and Ki67 þ, whereas some located adjacent to the forming dermal papilla begin to express tyrosinase-related protein-1, an early marker of di¡erentiation. Melanin gran-
ules appear in stage 5 hair follicles coincident with tyrosinase expression in nonproliferating tyrosinase-related protein-2 þ /tyrosinase-related protein-1 þ melanocytes. Stage 6^8 hair follicles, those actively producing hair, show nonproliferating tyrosinase-related protein2 þ melanocytes in the bulge area, tyrosinase-related protein-2 þ /tyrosinase-related protein-1 þ melanocytes in the outer root sheath, and tyrosinase-related protein-2 þ /tyrosinase-related protein-1 þ /tyrosinase þ melanocytes above the dermal papilla. These data suggest that melanocyte precursor cells proliferate extensively at the onset of follicle development. Progeny of these cells migrate down the developing follicle, differentiating further until reaching the area immediately above the dermal papilla, where fully di¡erentiated nonproliferative melanin-producing melanocytes persist, contributing pigment to the growing hair shaft. Key words: morphogenesis/skin neuralcrest/melanin. JID Symposium Proceedings 8:76 ^79, 2003
H
Melanogenesis is controlled by several key enzymes that are uniquely expressed in melanocytes (reviewed in Hearing, 1999). Tyrosinase catalyzes the rate-limiting initial events of melanogenesis, and mutations in tyrosinase gene lead to loss of pigment (Beermann et al, 1990). Tyrosinase-related proteins (TRP) 1 and TRP2 share 40^45% amino acid identity with tyrosinase and are also critically important for melanogenesis, functioning as downstream enzymes in the melanin biosynthetic pathway (Winder et al, 1994); and mutations in these genes also a¡ect pigmentation in mice. In postnatal murine anagen HF, melanocytes are divided into three distinct subpopulations. The ¢rst is located in the HF bulge, expresses only TRP2, does not proliferate, and presumptively represents melanocyte stem cells. The second is located in the HF outer root sheath, expresses TRP2 and relatively weak TRP1, displays proliferative activity, and represents di¡erentiating melanocytes. The third is located in the hair matrix above the DP, expresses TRP2, TRP1, and tyrosinase, does not proliferate, and actively produces melanin (Tobin et al, 1999; Botchkareva et al, 2001). The dynamics of the formation of HF pigmentary units in developing HF is only poorly understood, however. As shown by immunohistochemical and transgenic studies, melanocytes in the developing HF express TRP2 and can be identi¢ed as early as stage 2 HF (Yoshida et al, 1996; Jordan and Jackson, 2000). It is unknown whether these melanocytes proliferate during transition to more di¡erentiated forms, and whether melanogenic
air follicle (HF) development is driven by inductive interactions between epidermal keratinocytes committed to hair-speci¢c di¡erentiation, and a specialized population of dermal ¢broblasts, which form a condensation beneath the hair placode and subsequently develop into follicular dermal papilla (DP) (reviewed in Philpott and Paus, 1998, Paus, 1998). During HF morphogenesis, neural crest-derived melanocytes migrate into the HF, produce and transport melanin to the keratinocytes of the precortical zone that di¡erentiate to form the pigmented hair shaft (reviewed in Starico, 1960, Hirobe, 1995, Lecoin et al, 1999). In developing HF, melanocyte migration and melanogenic activity are controlled by stem cell factor and its receptor c-kit. Loss of function for these proteins either in corresponding mouse mutants or after treatment with c-kit neutralizing antibody results in the formation of nonpigmented hairs (Chabot et al, 1988, Zsebo et al, 1990, Nishikawa et al, 1991, Yoshida et al, 1996, Jordan and Jackson, 2000).
Manuscript accepted for publication February 1, 2003 Reprint requests to: Barbara A. Gilchrest, Department of Dermatology, Boston University School of Medicine, 609 Albany Street, Boston, MA 02118, U.S.A. Email: [email protected] 1 Present address: The Gillette Company, Needham, MA, U.S.A. Abbreviations: HF, hair follicle; DP, dermal papilla; TRP, tyrosinaserelated protein.
0022-202X/03/$15.00 . Copyright r 2003 by The Society for Investigative Dermatology, Inc. 76
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Figure 1. Expression patterns for melanogenic proteins during hair follicle morphogenesis. Cryosections of back skin of E16.5^18.5 or P0.5^6.5 C57BL/6 mice were immunostained with anti-sera against proliferative marker Ki- 67 (red £uorescence) and one of the melanogenic proteins (TRP1,TRP2, or tyrosinase; green £uorescence). Slides were counterstained with TO-PRO-3 (blue £uorescence). Left column of ¢gures shows schematic drawings of distinct stages of hair follicle development (Paus et al, 1999). (A) Colocalization of Ki- 67 and TRP2 (arrow) in undi¡erentiated melanocytes of stage 1 HF. Dermal^epidermal border is indicated by dotted line. (B) Colocalization of Ki- 67 and TRP2 (arrows) in undi¡erentiated melanocytes of stage 3 HF. (C) Lack of Ki- 67 in TRP1 þ di¡erentiating melanocytes (arrow) of stage 3 HF. (D) Colocalization of Ki- 67 and TRP2 in melanocytes located in central portion of stage 4 HF (arrow). Lack of Ki- 67 in TRP2 þ melanocytes located close to the DP (arrowhead) of stage 5 HF. (E) Lack of Ki- 67 in TRP1 þ melanocytes located close to the DP (arrowhead) of stage 5 HF. (F) Hair bulb melanocytes of fully developed stage 8 HF express tyrosinase and are Ki- 67 (arrows).
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BOTCHKAREVA ET AL
JID SYMPOSIUM PROCEEDINGS
activity of HF melanocytes detected at more advanced stages of HF morphogenesis (Hirobe, 1995, Paus et al, 1999) is associated with changes in expression of other melanogenic proteins (TRP1, tyrosinase). We have now systematically analyzed the expression of TRP1, TRP2, and tyrosinase in proliferating, di¡erentiating, and melanogenically active melanocytes during HF development in normal C57BL/6 mice. This study provides evidence that HF development and formation of the HF pigmentary unit are highly synchronized, and every distinct stage of HF development is associated with its own patterns of melanocyte proliferation, di¡erentiation, and melanogenic activity. MATERIALS AND METHODS Animals and tissue collection C57BL/6 mice were purchased from Charles River (Cambridge, MA) and housed in community cages at the animal facility of the Boston University School of Medicine. For the analysis of HF morphogenesis, embryonic and neonatal skin was harvested at various time points of estimated gestational age [embryonic days (E) 14.5, E16.5, and E18.5], or postnatal age [postnatal days (P) 0, P1, P2, P3, P6, P10 after birth]. For immunohistochemical analysis, the back skin was harvested parallel to the vertebral line embedded rapidly and snap-frozen in liquid nitrogen, using a special technique for obtaining longitudinal cryosections through the HF from one de¢ned site (Paus et al, 1999). Immunohistochemistry and multicolor confocal microscopy For immunohistochemical analyses, acetone-¢xed cryostat sections (8 mm) of adolescent C57BL/6 mouse back skin were used. To study the expression of tyrosinase, TRP1, or TRP2, we performed an immuno£uorescence protocol using the corresponding primary rabbit anti-sera (gift of Dr V. Hearing) and secondary tetramethylrhodamine isothiocyanate-conjugated goat-anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA), as described previously (Botchkareva et al, 2001). For double immunovisualization of melanogenic proteins and the proliferative marker Ki- 67, cryosections were ¢rst incubated with rat monoclonal antibody against murine Ki- 67 (Dianova, Hamburg, Germany), following by incubation with tetramethylrhodamine isothiocyanate-labeled goat anti-rat IgG (Jackson ImmunoResearch). After subsequent washing in Tris-bu¡ered saline, sections were incubated with primary rabbit anti-serum against one of the melanogenic proteins (TRP1, TRP2, or tyrosinase) followed by incubation with £uorescein isothiocyanate-conjugated secondary goatanti-rabbit rat IgG (Jackson ImmunoResearch). Finally, sections were counterstained by TO-PRO3 -iodide (Molecular Probes Inc., Eugene, OR) for visualization of cell nuclei. All slides were analyzed using a Zeiss confocal microscope, and were photo-documented using a digital image analysis system (Pixera, San-Diego, CA).
RESULTS AND DISCUSSION
To de¢ne distribution and proliferative activity of melanocytes during formation of the hair pigmentary unit, we performed double immuno-visualization of melanogenic proteins (TRP1, TRP2, tyrosinase) and the proliferative marker Ki- 67 in cryosections of C57BL/6 murine embryonic (E14.5^18.5) and neonatal (P0.5^10) skin. We found that TRP2 þ and Ki- 67 þ melanocytes were already seen in stage 1 HF as located among keratinocytes of the hair placode (Fig 1A); however, those melanocytes were TRP1^ and tyrosinase^ (not shown), consistent with minimal di¡erentiation. In stage 3 HF, many TRP2 þ melanocytes were visible in the elongating HF and showed Ki- 67 expression (Fig 1B), consistent with migration of these cells down the developing follicle. Single TRP2 þ and Ki- 67^ cells were also seen in stage 3 HF (not shown). TRP1 expression was ¢rst detected in single cells of stage 3 HF, most of them were Ki- 67^ (Fig 1C), consistent with progressive melanocyte di¡erentiation. In stage 5 HF, numerous TRP2 þ and TRP1 þ melanocytes were located immediately above the DP and did not show Ki- 67 expression (Fig 1D,E). Single melanocytes located here were also tyrosinase þ and Ki- 67^ (not shown). During HF progression to the latest stages of development (stages 6^8), tyrosinase expression in the melanocytes located above DP was strongly enhanced
Figure 2. Fate of melanocytes during morphogenesis of hair follicle pigmentary unit. TRP2 þ melanocytes precursor cells (melanocytes stem cells) proliferate extensively at the onset of follicle development. Di¡erentiation of their progeny is associated with inhibition of proliferation and the onset of TRP1 expression. Fully di¡erentiated nonproliferative melanin-producing melanocytes that contribute pigment to the growing hair shaft express all three melanogenic proteins.
(Fig 1F). These melanocytes were also TRP2 þ and TRP1 þ and Ki- 67^, consistent with full di¡erentiation; however, no tyrosinase expression was seen in single TRP2 þ and TRP1 þ melanocytes located in the HF outer root sheath (not shown). Our data and reports published previously (Yoshida et al, 1996, Jordan and Jackson, 2000) suggest that the genetic program of formation of the hair pigmentary unit is organized as a multistep transition of undi¡erentiated melanocytes progenitors to fully di¡erentiated cells actively producing and transporting melanin to hair shaft keratinocytes (Fig 2). As we showed here (Fig 1), this process is highly co-ordinated with distinct morphogenic steps of HF development. Most likely, HF melanocytes arise from the TRP2 þ progenitors that are located in embryonic epidermis (Nishikawa et al, 1991, Yoshida et al, 1996; Jordan and Jackson, 2000). These presumptive stem cells then migrate into developing hair placode (stages 1^2 of HF morphogenesis). At this stage they express TRP2, but do not express the di¡erentiation markers TRP1 and tyrosinase, and proliferate (Figs 1 and 2). Transition of TRP2 þ melanocytes to a more di¡erentiated status is associated with the onset of TRP1 expression and with reduction of their proliferation, as it is visible at stage 3 HF (Fig 1). The onset of melanogenic activity, which is detected in those melanocytes that are closely adjacent to the DP of stage 5 HF (Hirobe, 1995, Paus et al, 1999), is accompanied with the appearance of tyrosinase expression (Figs 1 and 2). Interestingly, tyrosinase expression and melanogenic activity is only detected in the subpopulation of HF melanocytes closest to the DP. This suggest that DP-secreted growth factors, such as stem cell factor, hepatocyte growth factor, and Wnt, are involved in the transition of HF melanocytes to fully di¡erentiated forms that actively produce melanin (Lindner et al, 1999, Botchkareva et al, 2001; Reddy et al, 2001). Therefore, this study provides important basic information about spatiotemporal organization of the HF pigmentary unit during HF development. These data also suggest that formation of the HF pigmentary unit is tightly coupled to distinct morphogenetic steps of HF development and that genetic programs of di¡erentiation of follicular keratinocytes, melanocytes, and DP ¢broblasts are highly synchronized. Molecular mechanisms that are involved in interactions between cells of HF epithelium and mesenchyme with follicular melanocytes during HF development, however, remain to be elucidated.
VOL. 8, NO. 1 JUNE 2003
DEVELOPMENT OF HAIR FOLLICLE PIGMENTARY UNIT
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