ARTICLE |
Correspondence to: Ralf Paus, Dept. of Dermatology, Charité, Humboldt-Universität zu Berlin, Schumannstr. 20/21, D-10117 Berlin, Germany. E-mail: ralf.paus@charite.de.
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Summary |
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Hair follicle development, growth (anagen), and regression (catagen) largely result from bidirectional epithelialmesenchymal interactions whose molecular basis is still unclear. Because adhesion molecules are critically involved in pattern formation and because the fundamental importance of neural cell adhesion molecule (NCAM) for feather development has been demonstrated, we studied the protein expression patterns of NCAM during hair follicle development and regression in the C57BL/6 mouse model. During murine hair follicle development, NCAM immunoreactivity (IR) was first detected on epithelial hair placodes and later on selected keratinocytes in the distal outer root sheath. Mesenchymal NCAM immunoreactivity (IR) was noted on fibroblasts of the future dermal papilla (DP) and the perifollicular connective tissue sheath. Fetal hair follicle elongation coincided with strong, ubiquitous dermal NCAM IR, which remained strong until the follicles entered into their first neonatal catagen. At this time, the strong interfollicular dermal NCAM IR decreased substantially. During consecutive hair cycles, mesenchymal NCAM IR was seen exclusively on DP and perifollicular connective tissue sheath fibroblasts and on the trailing cells of regressing catagen hair follicles. These highly restricted and developmentally controlled expression patterns suggest an important role for NCAM in hair follicle topobiology during morphogenesis and cyclic remodeling of this miniorgan. (J Histochem Cytochem 46:14011409, 1998)
Key Words: adhesion molecules, connective tissue sheath, dermal papilla, fibroblasts, catagen
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Introduction |
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The bidirectional interactions between dermal papilla (DP) fibroblasts of the hair follicle and follicular keratinocytes that drive hair follicle development and growth offer an intriguing model for dissecting inductive processes, not only during morphogenesis but also in the adult mammal (
For several reasons, neural cell adhesion molecule (NCAM) is a particularly interesting candidate as a key adhesion molecule in hair follicle topobiology (
During feather follicle morphogenesis, NCAM-mediated cell-cell interactions are crucial to the appropriate formation of the dermal condensations that are the avian analogue of the developing mammalian DP. The application of anti-NCAM antibodies during feather follicle morphogenesis in vitro induced a highly irregular shape of the dermal condensations (
Given the puzzling differences in NCAM immunoreactivity patterns that have been described in skin (
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Materials and Methods |
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Animal Models and Tissue Collection
Syngenic C57BL/6 mice (female, 121-day-old neonatal or 69-week-old; Charles River, Sulzfeld, Germany) were housed in community cages, with 12-hr light periods, at the Charitè animal facilities, Virchow Campus, Berlin, and were fed water and mouse chow ad libitum. Active hair growth (anagen) was induced in the back skin of adolescent mice in the telogen phase of the hair cycle (as judged from their homogeneously pink back skin color) by application of a melted wax/resin mixture under general anesthesia as described (
The neck region of the back skin was harvested parallel to the vertebral line to obtain longitudinal sections through the hair follicles from one defined site as described (
Immunohistochemistry
Ten-µm sections were used to analyze fetal skin and 8-µm sections were used for neonatal and adolescent skin. The sections were incubated with a monoclonal rat anti-mouse antibody (clone H28-123-16; Boehringer) against the extracellular domain of NCAM specifically binding to NCAM 120, 140, and 180 (
Antigenantibody complexes were visualized by both immunofluorescence (IF) and avidinbiotincomplex (ABC) labeling. For the ABC labeling, cryostat sections were incubated with the primary antibody for 1 hr at room temperature (RT) (1400), followed by an incubation with biotin-conjugated F(ab)2 fragments of a goat anti-rat IgG (Jackson Immunoresearch, Bar Harbor, ME; 1200, 30 min, RT). Skin sections were then incubated with ABC complex (Vecta-Stain Kit; Vector Laboratories, Burlingame, CA) for 45 min at RT. The alkaline phosphatase color reaction was developed as described (
Immunomorphometry
The immunoreactivity (IR) pattern was scrutinized by studying at least 20 microscopic fields or hair follicles per mouse, and five mice were assessed per stage of hair follicle morphogenesis and cycling. Thus, we examined at least 100 microscopic fields or hair follicles derived from at least five different mice per stage of hair follicle morphogenesis and cycling. The most consistently recognizable specific IR patterns were recorded and summarized in schematic representations of murine hair follicle morphogenesis and cycling that had been computer-generated (
Because we have previously described hair cycle-dependent NCAM expression patterns in selected skin nerve fibers and Schwann cells (
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Results |
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The expression patterns of NCAM in skin epithelium and mesenchyme during fetal and neonatal hair follicle morphogenesis and during later hair follicle cycling were strikingly different and appeared to be subject to stringent developmental controls. Figure 1 shows representative NCAM IR patterns during various stages of hair follicle morphogenesis, regression and consecutive cycling, which are summarized schematically in Figure 2.
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NCAM Expression During Fetal Hair Follicle Morphogenesis
In late fetal skin at E16, no cutaneous NCAM IR was found during Stage 1 of fetal tylotrich hair follicle morphogenesis in C57BL/6 mice. The first epithelial NCAM IR was detected in the hair placode epithelium of fetal back skin during Stage 2 (Figure 1A). No (or only extremely weak) mesenchymal NCAM IR was detected on the underlying dermal fibroblast condensations. At E18 (Stages 35), NCAM IR became restricted to a selected group of keratinocytes of the developing ORS, and the developing DP and the fibroblasts of the distal perifollicular connective tissue sheath showed increasing NCAM IR. Intermediate NCAM IR was found throughout the dermis (Stage 4; Figure 1B). NCAM IR remained strong throughout hair follicle development (Stages 58) on keratinocytes in the peri- and suprainfundibular region of the ORS (Figure 1B and Figure 2). During Stages 58, the dermal skin compartment showed increased homogeneous NCAM IR compared to the preceding stages, which was particularly accentuated on DP fibroblasts (Figure 1C and Figure 2). Throughout fetal hair follicle development, heterogeneous NCAM staining patterns were noted on the arrector pili muscle, but no NCAM IR was noted in the sebaceous gland (Figure 1H and Figure 1I).
NCAM Expression During Neonatal Hair Follicle Morphogenesis
Comparing fetal (Figure 1A, Figure 1B, and Figure 2, Stages 15, left) and neonatal hair follicle development (Figure 1CF and Figure 2, Stages 15, right) in mice, we found much more intense NCAM IR in neonatal skin compared to fetal skin, yet largely the same follicular NCAM IR patterns, whereas a significant change in the dermal expression patterns of NCAM was noted. In contrast to hair follicle development in fetal skin, NCAM was strongly and homogeneously expressed throughout the dermis during neonatal hair follicle morphogenesis (Figure 1CF and Figure 2, Stages 15, right). Neonatal hair follicle elongation (Stages 58) was associated with strongly pronounced NCAM IR on the proximal perifollicular connective tissue sheath and on DP fibroblasts (Figure 1C, Figure 1E, and Figure 1F). Similar to the findings in fetal skin, strong NCAM IR was noted on selected keratinocytes in the distal isthmus region of the developing ORS during Stages 3 and 4 (Figure 1D) and in the peri- and suprainfundibular ORS during Stages 58 (Figure 1C and Figure 1E), while heterogeneous NCAM IR patterns were noted on the arrector pili muscle. Again, sebocytes did not display any NCAM IR.
NCAM Expression During the First Neonatal Catagen and the Beginning of Hair Follicle Cycling
During the first neonatal hair follicle regression (catagen) (Figure 1G), the strong interfollicular dermal NCAM IR declined dramatically (Figure 1GI). The regressing epithelial strand showed intermediate NCAM IR (Figure 1G), and the fibroblasts of the proximal perifollicular connective tissue sheath, as well as the trailing mesenchymal cells, displayed intense NCAM IR (Figure 1G). During the initiation of the first neonatal hair cycle (Figure 1H), interfollicular dermal NCAM IR was very weak compared to neonatal hair follicle morphogenesis. This dermal NCAM expression pattern was maintained throughout consecutive hair cycles (Figure 1I). In contrast to the interfollicular dermis, the perifollicular connective tissue sheath and DP fibroblasts of the first anagen I hair follicles showed intense NCAM IR (Figure 1H).
NCAM IR of periinfundibular ORS keratinocytes was observed to be particularly pronounced on tylotrich hair follicles (Figure 1J) in a strikingly hair cycle-dependent manner. Intense periinfundibular ORS NCAM IR has been found throughout neonatal hair follicle morphogenesis (Figure 1J) and middle anagen (Figure 1I), which decreased strongly during catagen (Figure 1K).
NCAM Expression During Catagen in Adolescent Skin
In contrast to fetal and neonatal hair follicle morphogenesis, during hair follicle regression in the skin of adolescent mice (Figure 1LO) mesenchymal NCAM IR was seen exclusively on DP and perifollicular connective tissue sheath fibroblasts and on the trailing cells of the regressing epithelial hair bulbs. Also in contrast to hair follicle morphogenesis, epithelial NCAM IR was seen on hair matrix keratinocytes during early catagen (Figure 1L) and on keratinocytes of the epithelial strand (Figure 1N and Figure 1O).
During late anagen VI and catagen III, the perifollicular connective tissue sheath was restricted to one layer of NCAM+ fibroblasts (Figure 1L). When the DP was moving upwards, the DP and the developing "tail" of trailing connective tissue sheath fibroblasts displayed very intense NCAM IR (Figure 1LO). With progressing catagen, the NCAM IR found on all hair matrix keratinocytes (Figure 1L and Figure 1M) became restricted to one layer of basement membrane-associated keratinocytes during late catagen (Figure 1N and Figure 1O). Heterogeneous NCAM IR patterns were detected on the arrector pili muscle throughout the entire hair cycle.
In summary, during fetal hair follicle morphogenesis, we noted a switch of NCAM expression from the hair placode epithelium to a ubiquitous and homogeneous NCAM expression pattern throughout the skin mesenchyme, with maximal NCAM expression by DP and perifollicular connective tissue sheath fibroblasts. During the first neonatal catagen, this interfollicular dermal NCAM IR declined dramatically. During the consecutive hair cycles, mesenchymal NCAM became highly restricted to the DP and the perifollicular connective tissue sheath. During neonatal and adolescent catagen, the trailing cells of the regressing epithelial hair bulb expressed the most intense NCAM IR.
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Discussion |
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In this report we present a profile of the developmentally controlled expression of NCAM on distinct fibroblast populations during fetal and neonatal morphogenesis of the skin and hair follicles, and define NCAM expression during the first neonatal catagen and the beginning of hair follicle cycling (Figure 2). In addition, we show that NCAM is also expressed in the cutaneous mesenchymal epithelium of murine skin, i.e., on selected keratinocytes of the hair placode and the developing hair bulb, as well as on ORS keratinocytes of anagen hair follicles and hair matrix keratinocytes of early catagen hair follicles (Figure 1).
Important previous studies in murine skin have focused mainly on the fetal development of tylotrich follicles (
Comparing fetal (Figure 1A, Figure 1B, and Figure 2, Stages 15, left) and neonatal hair follicle development in mice (Figure 1CF and Figure 2, Stages 15, right), we found much more intense NCAM IR in neonatal skin compared to fetal skin, i.e., a significant change in the dermal expression patterns of NCAM was noted. In contrast to hair follicle development in fetal skin, NCAM was strongly and homogeneously expressed throughout the dermis during neonatal hair follicle morphogenesis (Figure 1CF and Figure 2, Stages 15, right). However, the follicular NCAM IR patterns were largely the same.
This peak of interfollicular dermal NCAM expression is seen only throughout the neonatal period of pelage hair follicle development (Figure 1DF). During fetal human hair follicle morphogenesis (
Because we have recently demonstrated strong expression of neurotrophins and their receptors during hair follicle morphogenesis and catagen development (
The functional importance of NCAM for skin appendage formation was first established by analyzing the development of feather follicles in chicken skin explant cultures (
As discussed elsewhere in greater detail (
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Acknowledgments |
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Supported in part by grants from the Deutsche Forschungsgemeinschaft (Pa 345/6-1, 8-1) and by Wella AG, Darmstadt, to RP.
The excellent technical assistance of R. Pliet, E. Hagen, and C. van der Veen and the help of M. Ünalan with the preparation of Figure 2 are most gratefully acknowledged.
Received for publication April 9, 1998; accepted July 8, 1998.
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