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Changing Patterns of Localization of Putative Stem Cells in Developing Human Hair Follicles
Changing Patterns of Localization of Putative Stem Cells in Developing Human Hair Follicles Masashi Akiyama,*² Lynne T. Smith,³ and Hiroshi Shimizu§¶
*Department of Dermatology, Teikyo University School of Medicine, Ichihara Hospital, Chiba, Japan; ²Division of Dermatology, Kitasato Institute Hospital, Tokyo, Japan; ³Departments of Biological Structure and Medicine (Dermatology), University of Washington School of Medicine, Seattle, Washington, U.S.A.; §Department of Dermatology, Hokkaido University School of Medicine, Sapporo, Japan; ¶Department of Dermatology, Keio University School of Medicine, Tokyo, Japan
In rodents, the hair follicle stem cells lie in a wellde®ned bulge in the outer root sheath; however, the bulge as a stem cell site of human hair follicle epithelium is still controversial. Epidermal stem cells are thought to express high levels of b1 integrin and low levels of E-cadherin and b- and g-catenin. In order to clarify the ontogenic distribution of possible stem cells during hair follicle development, the expression patterns of b1 integrin subunits, E-cadherin, and band g-catenins in the skin samples from human fetuses of a series of estimated gestational ages (EGA) were examined. b1 integrin-rich, E-cadherin-, and b- and g-catenin-poor cells, possible stem cells, were localized to the entire hair germ (65±84 d EGA) and later to the outermost cells of hair peg (85±104 d EGA). In the bulbous hair peg (105±135 d EGA) and in the differentiated lanugo hair follicle (>135 d
EGA), they were settled in the bulge and the outermost layer of the outer root sheath. This sequential localization was similar to that of cells rich in epidermal growth factor receptor expression and positive with keratin 19, a putative marker of epidermal stem cells. In addition, these b1 integrin-rich, E-cadherin-, and b- and g-catenin-poor cells showed similar, undifferentiated morphologic features by electron microscopy. This information of ontogenic localization of possible hair follicle stem cells contributes to the further understanding of mechanisms of human hair follicle morphogenesis and supports the idea that the human fetal hair follicle bulge is a site of stem cells for follicular epithelium. Key words: cell adhesion molecule/electron microscopy/epidermal growth factor receptor/fetal development/keratin 19. J Invest Dermatol 114:321±327, 2000
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was mostly marked in the intermediate part and the lower half of the central part of plucked human hair follicles. Thus, there is a discrepancy between in vivo and in vitro ®ndings and the localization sites of stem cells of human developing hair follicle are still controversial. Integrins, surface receptors mediating cell±cell and cell±extracellular matrix adhesion relevant for a number of physiologic and pathologic phenomena (Albelda and Buck, 1990; Crossin, 1990; Hemler, 1990; Springer, 1990; Edelman and Crossin, 1991), regulate cell adhesion, shape, motility, intracellular signaling, and gene expression (Werb et al, 1989; Springer, 1990; Hynes, 1992). The b1 family of integrins, including a2b1 and a3b1 integrins on epithelial cells, are believed to function in cell±cell interaction, cellsubstrate adhesion (Symington et al, 1993; Larjava et al, 1990; Marchisio et al, 1991), signal transduction for extracellular matrix (Damsky and Werb, 1992; Miyamoto et al, 1995), cell±cell/cell± substrate adhesion (Carter et al, 1990; Marchisio et al, 1991), and cell fate (Jones and Watt, 1993; Symington et al, 1993) of human keratinocytes. Recently, experiments with cultured keratinocytes have established that b1 integrins, including a2b1 and a3b1 integrins, not only mediate cell adhesion and migration, but also regulate strati®cation and the initiation of epithelial differentiation and morphogenesis (Hertle et al, 1991; Adams and Watt, 1993). Furthermore, stem cells are thought to express higher levels of the a2b1 and a3b1 integrins in human epidermis than keratinocytes of lower proliferative potential (Jones and Watt, 1993; Jones et al, 1995; MoleÁs and Watt, 1997).
he hair follicle stem cells have been reported to lie in the bulge region of hair follicles in rodents (Cotsarelis et al, 1990). The human hair follicle bulge is also hypothesized as a stem cell site (Lavker et al, 1991, 1993; Sun et al, 1991). The ``bulge-activation hypothesis'', proposing that hair cycles re¯ected activation and inactivation of stem cells in the bulge, has been developed on the basis of the identi®cation of putative hair follicle stem cells in the bulge region (Cotsarelis et al, 1990; Sun et al, 1991). Recently, b1 integrin bright cells were identi®ed in adult human hair follicle bulges, as was keratin 15 (Lyle et al, 1998). In addition, label-retaining cells were found in the human hair follicle bulge and the human adult hair follicle bulge was strongly suggested to be the stem cell site for hair follicle epithelium (Lyle et al, 1998). From the results of in vitro studies, however, hair follicle stem cells are reported to be either close to the attachment site of the arrector pili muscle (Yang et al, 1993) or slightly lower down (Rochat et al, 1994), or in the follicular bulb (Reynolds et al, 1993) in adult human hair follicles. Moll (1995) reported that colony-forming ability
Manuscript received February 16, 1999; revised October 29, 1999; accepted for publication November 3, 1999. Reprint requests to: Dr. Masashi Akiyama, Department of Dermatology, Teikyo University School of Medicine, Ichihara Hospital, 3426±3, Anesaki, Ichihara, Chiba 299±0111, Japan. Email: [email protected] Abbreviations: EGA, estimated gestational age; K19, keratin 19. 0022-202X/00/$ 15.00
´ Copyright # 2000 by The Society for Investigative Dermatology, Inc. 321
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Cadherins were reported to play a major role in epithelial morphogenesis (Takeichi, 1995) and may regulate keratinocyte growth and differentiation (Zhu and Watt, 1996). Cadherinmediated adhesion is involved in the downregulation of integrin expression that occurs during keratinocyte terminal differentiation (Hodivala and Watt, 1994). The mechanism by which cadherins could regulate integrin expression is presently under investigation and is believed to involve the mutual association of integrins and cadherins with the actin cytoskeleton, which, in the case of cadherins, is mediated by catenins (Braga et al, 1995). There is clear evidence that b-catenin has signaling functions that may be independent of its role in cell±cell adhesion (Barth et al, 1997). In fact, inversely correlated expression of levels of b1 integrins and E-cadherin or b-catenin in basal keratinocytes of the interfollicular epidermis were reported (MoleÁs and Watt, 1997). Thus, dull stainings for E-cadherin and b-catenin are thought to be a stem cell marker (Watt, 1998). Ontogenically, human hair follicle development starts as a hair germ [65±84 d estimated gestational age (EGA)], which is a bud from the epidermis into the dermis and is associated with an underlying condensation of mesenchymal cells (Holbrook and Odland, 1978; Holbrook, 1979; Holbrook et al, 1993). In the next stage of development, the hair peg (85±104 d EGA) forms as the cord of epidermal cells growing into the dermis. In the hair follicle of bulbous hair peg stage (105±135 d EGA), each part of the follicle begins to differentiate into regions that are de®ned by the position of outgrowths of outer root sheath cells including the sebaceous gland primordium and the bulge. The ¯at end of the hair peg molds into a bulb and the associated mesenchymal cells become the dermal papilla. Finally, differentiated hair follicles that generate the thin hair seen at birth named ``lanugo hair'' are formed in the late second trimester (>135 d EGA). In this study, we investigated the expression of b1, a2, and a3 integrins, E-cadherin, and b- and g-catenins in the developing human hair follicles of skin samples from human fetuses of a series of EGA (49±163 d EGA) in order to elucidate whether these cell adhesion molecules are involved in human hair follicle morphogenesis, and to clarify the stem cell sites in the early stage of developing human fetal hair follicles. In addition, we identi®ed b1 integrin-rich, E-cadherin-, and b- and g-catenin-poor cells and compared these cells with keratin 19 (K19)-positive, epidermal growth factor (EGF) receptor-rich cells, based on the evidence that K19 is a marker for epidermal stem cells (Lane et al, 1991; Michel et al, 1996) and that the stem cells strongly express EGF receptor (Akiyama et al, 1996). We also observed the ultrastructural features of these possible stem cells (b1 integrin-rich, E-cadherin-, and band g-catenin-poor) in human developing hair follicles. Our ®ndings demonstrated that possible stem cells for hair follicle epithelium were present in the entire hair germ, became localized ®rst to outermost cells of hair peg, and then in the bulge and the outermost layer of the outer root sheath (ORS). This sequential localization was similar to that of K19-positive, EGF receptor-rich cells. Ultrastructurally, these cells at any stage of hair follicle development showed similar, undifferentiated features that distinguished them from other follicular cells. These ®ndings of unique sequential localization of potential follicular stem cells are important for understanding the mechanisms of initiation, induction, and development of human fetal hair follicles. MATERIALS AND METHODS Tissue Human embryonic and fetal skin specimens were obtained from abortuses of 49±163 d EGA through the Central Laboratory of Human Embryology at the University of Washington, Seattle, U.S.A. with the approval of the Human Subjects Review Board and in accordance with the United States DHEW policies. The ages, the autopsy sites, and the numbers of embryos or fetuses included in this study were as follows: 49±64 d EGA, scalp (n = 2), trunk (n = 2); 65±84 d EGA, scalp (n = 3), trunk (n = 2); 85± 104 d EGA, scalp (n = 2), trunk (n = 2); 105±135 d EGA, scalp (n = 2), trunk (n = 2); >135 d EGA, scalp (n = 2), trunk (n = 2). Two or three skin specimens from each fetus were used for the study. EGA was determined
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from maternal histories, fetal measurements (crown rump and foot length), and comparative histologic appearance of epidermis (Shepard, 1975; Holbrook, 1979; Mercer et al, 1987). Antibodies The primary antibodies used in this study were mouse monoclonal antihuman a2 integrin antibody, Clone P1E6 (Carter et al, 1990) (DAKO, Carpinteria, CA), mouse monoclonal antihuman a3 integrin antibody, Clone P1B5 (Wayner and Carter, 1987) (DAKO), mouse monoclonal antihuman b1 integrin antibody, Clone DE9 (Bergelson et al, 1992) (Upstate Biotechnology, Lake Placid, NY), mouse monoclonal antihuman E-cadherin antibody, HECD-1 (Shimoyama et al, 1989), rabbit polyclonal antihuman E-cadherin antibody, Jelly (Damsky et al, 1983), mouse monoclonal anti-b catenin antibodies, Clone 14 (Transduction Laboratories, Lexington, KY) and 5H10, mouse monoclonal anti-g catenin antibody, 4F11, mouse monoclonal antihuman EGF receptor antibody, Clone EGFR1 (DAKO, Glostrup, Denmark), and mouse monoclonal antihuman K19 antibody, LP2K (Lane et al, 1985). Immuno¯uorescent labeling Fetal skin was quick frozen in a dry iceethanol slush and 6 mm thick sections were cut using a cryostat. At least three sections from each specimen were stained and examined. The sections were incubated in normal goat serum for 30 min and then incubated in primary antibody solution for 1 h at 37°C. Antibody dilutions were 1:50 for anti-a2 integrin antibody and anti-a3 integrin antibody, 1:40 for anti-b1 integrin antibody, 1:500 for HECD-1, 1:160 for Jelly and antib-catenin, clone 14, 1:5 for anti-EGF receptor antibody, and 1:10 for LP2K. Antibodies 5H10 and 4F11, were used neat. The sections were then incubated in ¯uorescein isothiocyanate (FITC)-conjugated to rabbit antimouse immunoglobulins (DAKO) or, for polyclonal antibody, Jelly, incubated in FITC-conjugated to goat antirabbit immunoglobulins (DAKO) for 30 min at room temperature, followed by 10 mg per ml propidium iodide to counterstain nuclei (Sigma, St. Louis, MO) for 10 s. The sections were extensively washed with phosphate-buffered saline between incubations. The stained sections were mounted with a cover slip in 50% glycerol mounting medium and stored in the refrigerator. Photos were taken with epi¯uorescent microscopy for several days after the immunostaining. Speci®c immunostainings were detected as green (FITC) and nuclear stain was observed as red (propidium iodide). Overlap of both FITC and propidium iodide was demonstrated as a yellowish color. Image analysis Image analysis for photo slides of a conventional immuno¯uorescence microscope was done using a image modi®cation software, Adobe Photoshop 3.0 (Adobe Systems, Mountain View, CA). In order to highlight the brightly stained cells, the strong signals of FITC (stronger than 20% of only green signals in histograms of distribution of signal level of pixels) were picked up from the images using a command, input-level adjustment. In order to reduce the contamination of propidium iodide ¯uorescence, all the red signals were weakened equally before the image analysis. A set exposure time was used for all the photo slides for image analysis. Electron microscopy Fetal skin was ®xed in one-half strength Karnovsky's ®xative or 2% glutaraldehyde solution, post®xed in 1% OsO4, dehydrated, and embedded in Epon 812 (Perry et al, 1987). All the samples were serially sectioned, sampled every 10±15 mm for light microscopy (1 mm thick), and thin sectioned for electron microscopy (70 nm thick). The histologic sections were stained by the method of Richardson et al (1960). The thin sections were stained with uranyl acetate and lead citrate (Reynolds, 1963) and examined under a JEOL 1200EXII transmission electron microscope in the transmission mode at 80 kV.
RESULTS b1 integrin-rich, E-cadherin-, and b- and g-catenin-poor cells were localized to the entire hair germ (65±84 d EGA) and the outer cells of the hair peg (85±104 d EGA) in the early development As for the results of all immunostainings, a consistent pattern was obtained in all the sections stained with each antibody at each developmental stage. Strong membranous immunostainings of b1, a2, and a3 integrin subunits were observed in the entire hair germ (Fig 1). At the same time, these b1 integrin-rich cells were E-cadherin- and b- and g-catenin-poor compared with suprabasal keratinocytes of the interfollicular epidermis. The outermost cells of hair peg showed strong membranous stainings of b1, a2, and a3 integrins (Fig 1). The outermost cells of hair peg were E-cadherin- and b- and g-catenin-
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Figure 1. b1 integrin-rich cells are seen in the entire hair germ, in the outer layer of the hair peg, and in the outermost layer of the early bulbous hair peg. Hair germ cells express g-catenin only weakly compared with those of interfollicular epidermis (a, b). b1 integrin is strongly expressed in the entire hair germ (c) and the outer cells of the hair peg (e). The expression of g-catenin is weak in the outer cells of the hair peg (d). a2 integrin is brightly expressed in the outermost cells of the early bulbous hair peg and the basal layer of the interfollicular epidermis (f). Speci®c immunostainings are demonstrated with ¯uorescein isothiocyanate (FITC, green) and nuclear stain is done with propidium iodide (red). Yellowish stain is caused mainly by the overlap of FITC ¯uorescence and propidium iodide ¯uorescence and partly by the nonspeci®c detection of FITC ¯uorescence by the ®lter for propidium iodide. (a±c) Hair germ (65±84 d EGA), (d, e) hair peg (85±104 d EGA), (f) early bulbous hair peg (108 d EGA), (c, e) b1 integrin, (f) a2 integrin, (a, b, d) g-catenin (4F11); scale bars: 50 mm.
poor compared with the inner cells of hair peg. The inner cells of hair peg were b1, a2, and a3 integrin-poor and E-cadherin- and b- and g-catenin-rich compared with the outermost cells. b1 integrin-rich, E-cadherin-, and b- and g-catenin-poor cells were speci®cally localized to the bulge and the outermost layer of ORS in the bulbous hair peg (105±135 d EGA) and the lanugo hair follicle (>135 d EGA) In the early bulbous hair peg, b1, a2, and a3 integrin-rich cells were localized to the outer cells all around the peg (Fig 1). These b1, a2, and a3 integrin-rich cells were E-cadherin-, b-, and g-catenin-dull compared with the inner cells (Fig 1). In the bulbous hair peg, bright membranous stainings of b1, a2, and a3 integrins were observed in the bulge and the outer cells of ORS. These b1 integrin-rich cells in the bulge and the outer cells of ORS were Ecadherin-, b-, and g-catenin-poor. In the differentiated lanugo hair follicle, bright staining of b1, a2, and a3 integrins was observed in the outer cells of the ORS and was weak in the inner cells of the ORS (Fig 2). a2 subunit is also brightly expressed in the matrix cells of the bulb. b1 and a3 integrins were only weakly expressed in the matrix cells of the bulb. In addition, the matrix cells of the bulb were strongly stained with b- and g-catenins, although the matrix cells were E-cadherin-poor. Thus, the b1 integrin-rich, E-cadherin-, b-, and g-catenin-poor cells were localized to the bulge and the outermost layer of the ORS both in the bulbous hair peg stage and in the lanugo hair follicle stage. Image modi®cation con®rmed the localization of b1 integrin-rich, E-cadherin-, b-, and g-catenin-poor cells in the bulge and the outermost layer of the ORS By the deletion of weak signals using image modi®cation, only the bulge cells and the outer layer cells of the ORS were revealed to exhibit
remaining strong signals for b1, a2, and a3 integrins (Fig 3). Image analysis revealed that the bulge region showed weak signals for Ecadherin and b- and g-catenins. These b1, a2, and a3 integrin-rich cells were E-cadherin-, b-, and g-catenin-poor. K19-positive cells and EGF receptor-rich cells showed similar ontogenic localization patterns to that of b1 integrin-rich, E-cadherin-, b-, and g-catenin-poor cells K19-positive cells were sequentially restricted to the hair germ, to the outer layer of hair peg, to the bulge and the outermost layer of the ORS in the bulbous hair peg and the lanugo hair follicles (Fig 4a). Cells in the hair germ and the outer layer of hair peg expressed EGF receptor strongly. Later, intense immunoreactivity of EGF receptor was seen in the cells in the bulge and the outermost layer of the ORS in the bulbous hair peg and the lanugo hair follicles (Fig 4b). The cells in the sites of ontogenic localization of b1 integrinrich, E-cadherin-, b-, and g-catenin-poor cells showed uniformly undifferentiated morphology Ultrastructurally, hair germ cells and cells in the outer layer of hair peg exhibited similar, undifferentiated features, including abundance of free ribosomes and glycogen particles in the cytoplasm and reduced number of cytoplasmic organelles indicative of differentiation, e.g., rough endoplasmic reticulum and intermediate ®laments (Fig 5a, b). In the bulbous hair peg and the lanugo hair follicle, bulge cells and cells in the outermost layer of the ORS showed similar, undifferentiated features to those seen in the cells in hair germ and the outer layer of the hair peg ultrastructurally (Fig 5c, d). Especially, the bulge consists of uniform, small undifferentiated cells.
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Figure 2. b1 integrin-rich, E-cadherin-, b-catenin-, g-catenin-poor cells are seen in the outermost layer of the ORS, but not in the matrix of the bulb in the lower portion of differentiated lanugo hair follicle (>135 d EGA). Bright stainings of b1 integrin (a), a2 integrin (b), and a3 integrin (c) are observed in the outermost layer (arrowheads) of the ORS. The outermost layer (arrowheads) of the ORS expresses Ecadherin (d), b-catenin (e), and g-catenin (f) very weakly. The matrix cells of the bulb are strongly stained with a2 integrin (b) and weakly stained with E-cadherin (d); however, in contrast to the outermost layer of the ORS, the matrix cells of the bulb express b1 integrin (a) and a3 integrin (c) only weakly and are brightly stained with b-catenin (e) and g-catenin (f). Speci®c immunostainings are demonstrated with FITC (green) and nuclear stain is done with propidium iodide (red). (a) b1 integrin, (b) a2 integrin, (c) a3 integrin, (d) E-cadherin (HECD-1), (e) b-catenin (Clone 14), (f) g-catenin (4F11); scale bar: 50 mm.
Possible stem cells were localized to the entire hair germ, and later to the outer cells of the hair peg, and were settled in the outermost layer of the ORS and the bulge of the bulbous hair peg and the lanugo hair follicle Summarizing the data shown above, possible stem cells, which were b1 integrinrich, E-cadherin- and b-catenin-poor, K19-positive and EGF receptor-rich, and undifferentiated in morphology, were sequentially localized to the entire hair germ and to the outermost cells of hair peg. Later in the bulbous hair peg stage and in the lanugo hair follicle stage, these possible stem cells were seen restrictively in the bulge and the outermost layer of the ORS (Fig 6). DISCUSSION In this study, unique sequential expression of b1, a2, and a3 integrin subunits, E-cadherin, and b- and g-catenins were shown at each stage of human hair follicle development. According to the results, strong stainings of b1, a2, and a3 integrins were seen in the entire hair germ (65±84 d EGA) and in the outermost cells of hair peg (85±104 d EGA) of the early stage of human hair follicle development. These results imply that cell±cell interaction and cell±extracellular matrix adhesion mediated by a2b1 and a3b1 integrins may be involved in early induction of human hair follicle morphogenesis. Based on the plural evidence that the b1 integrin-rich, Ecadherin-and b- and g-catenin-poor cells are stem cells of keratinocytes (MoleÁs and Watt, 1997), these results indicated that the entire hair germ consists of possible pluripotent stem cells and, in the next stage, that the possible stem cells are restricted only to the outer layer in the hair peg. Finally in the late stage of follicular development, possible stem cells of the hair follicle epithelium are thought to be localized in the bulge and the outermost layer of the
ORS (Fig 6). Our ®ndings of the dynamic ontogenic localization of possible stem cells provide important clues to understanding the mechanism of human hair follicle development. In developing hair follicles in human fetuses, the bulge is a prominent hemispherical protrusion (Madsen, 1964) consisting of undifferentiated cells that are immunolabeled with an antibody to K19 (Lane et al, 1985; Akiyama et al, 1995), a possible stem cell marker (Lane et al, 1991; Michel et al, 1996). Keratin 15-positive, b1 integrin bright cells were identi®ed in adult human hair follicle bulges (Lyle et al, 1998). In addition, label-retaining cells were found in the adult human hair follicle bulge (Lyle et al, 1998). The human fetal bulge cells are also known to strongly express EGF receptor (Akiyama et al, 1996). These observations were con®rmed in this study. The existence of K19 keratin and strong expression of EGF receptor in the bulge cells may support the notion that the bulge is a hair follicle stem cell site also in human. Our observations show that cells in the bulge are unique in that they are uniformly b1 integrin-rich, E-cadherin-, and b- and g-catenin-poor. These facts further support the notion that the bulge is a stem cell site of hair follicle epithelium at least during the fetal period. In the epidermis of human skin, expression of the b1 integrin in vivo and in culture is con®ned to the basal cells in contact with basement membrane (Kaufman et al, 1989; De Luca et al, 1990; Hertle et al, 1991; Cerri et al, 1994). In adult human anagen hair follicles, a shift in expression of a2b1 and a3b1 integrins by the outer ORS cells from a basolateral distribution (basement membrane zone) in the lower ORS to an apicolateral expression (nonbasal side) in the upper ORS was reported (Commo and Bernard, 1997). These altered localization patterns may re¯ect the two different functions of a2b1 and a3b1 integrins, i.e., cell±cell adhesion and cell±extracellular matrix interaction. Interestingly, in this study, all cells in the fetal bulge including the inner cells, which are not in contact with the extracellular matrix, showed strong
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Figure 3. The modi®ed images clearly demonstrate that the bulge of the lanugo hair follicle (>135 d EGA) consists of b1 integrinrich, E-cadherin-, b-catenin-, g-catenin-poor cells. Deletion of the weak signals from the immuno¯uorescent images of the mid-portion of the lanugo hair follicle revealed that the strong signals of b1 integrin (a), a2 integrin (b), and a3 integrin (c) remain in the bulge (arrowheads), although cells in the bulge (arrowheads) show no strong signals of E-cadherin (d), b-catenin (e), and g-catenin (f). Speci®c immunostainings are demonstrated with FITC (green) and nuclear stain is done with propidium iodide (red). (a) b1 integrin, (b) a2 integrin, (c) a3 integrin, (d) E-cadherin (HECD-1), (e) b-catenin (5H10), (f) g-catenin (4F11); scale bar: 50 mm.
stainings for b1, a2, and a3 integrin subunits. In addition, the fetal bulge cells exhibited poor expression of E-cadherin and b- and gcatenins. These facts suggest that the fetal bulge cells are a uniform and unique population in terms of expression of cell adhesion molecules on the cell surface. Carroll et al (1995) established b1 integrin transgenic mice using the involucrin promoter that express functional human b1 integrin in the suprabasal epidermal layers. The b1 integrin transgenic mice showed abnormalities of the hairs of the coat and whiskers. The abnormal coat phenotype was apparent as soon as the ®rst coat developed, and there was a progressive normalization through successive hair growth cycles. These ®ndings suggest that regulated b1 integrin expression is essential for the hair follicle development, although the exact mechanisms of functions of b1 integrins for the hair follicle morphogenesis are still unknown. In this context, our ®ndings of ontogenic localization of b1 integrin expression is important to elucidate the roles of b1 integrins in human hair follicle development. In this study, the selected criteria for follicle stem cell stainings, b1 integrins high, E-cadherin-low, b- and g-catenins-low, were extrapolated from work with epidermal stem cell (MoleÁs and Watt, 1997). Our results not only demonstrated the patterns of localization of putative stem cells in human developing hair follicle, but also revealed that the stainings for E-cadherin, b- and g-catenins are useful markers for stem cells of follicular epithelium as well as epidermal stem cells, at least in the fetal period. Existence of a population of skin pluripotent stem cells has been hypothesized (Cotsarelis et al, 1990). In the hypothesis, the pluripotent stem cells are thought to provide transient amplifying cells not only to hair follicles, but also to the epidermis. These
Figure 4. K19-positive, EGF receptor-rich cells are also seen in the bulge and the outermost layer of the lanugo hair follicle. (a) In the lanugo hair follicle (136 d EGA), cells in the bulge (arrows) and in the outermost layer of the ORS (arrowheads) are K19-positive. (b) Cells in the bulge (arrows) and in the outermost layer of the ORS (arrowheads) show stronger immunoreactivity for EGF receptor than the other parts of the developing hair follicle. (a) K19 staining; (b) EGF receptor staining; arrows, bulge; arrowheads, outermost layer of ORS; scale bar: 50 mm.
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Figure 5. Cells in the hair germ, the outer layer of hair peg, and the bulge and the outermost layer of the bulbous hair peg show similar, undifferentiated morphologic features. Electron microscopy reveals that hair germ cells (a), cells in the outer layer of hair peg (b), cells in the bulge of bulbous hair peg (c), and cells in the outermost layer of the ORS of bulbous hair peg (d) show a reduced amount of intermediate ®lament bundles and rough endoplasmic reticulum, and abundance of glycogen particles and free ribosomes in the cytoplasm. The nucleus/cytoplasm ratio of these cells is high and chromatin aggregation in the nuclei appears poor. (a) Cells in the hair germ (69 d EGA); (b) cells in the outer layer of hair peg (89 d EGA); (c) cells in the bulge of bulbous hair peg (125 d EGA); (d) cells in the outermost layer of the ORS of bulbous hair peg (125 d EGA); scale bars: 1 mm.
Figure 6. Sequential localization of b1 integrin-rich, E-cadherin-, b-catenin-, g-cateninpoor cells during human fetal hair follicle development. Ontogenically, b1 integrin-rich, Ecadherin-, and b- and g-catenin-poor cells, possible hair follicle stem cells, are restricted to the entire hair germ, the outer cells of hair peg, then the bulge and the outer layer cells of the bulbous hair peg, and ®nally the bulge and the outermost cells of the ORS of lanugo hair follicle. Dotted area, restriction sites of b1 integrin-rich, E-cadherin-, and b- and g-catenin-poor cells; arrows, the bulge.
results demonstrated that stem cells of hair follicle epithelium and those of epidermal keratinocytes have similar features of expression to cell adhesion molecules and this fact may support the idea of the existence of a skin pluripotent stem cell population shared by the hair follicle epithelium and the epidermal keratinocytes. On the other hand, there is a possibility that the characteristic expression pattern of adhesion molecules is not speci®c to stem cells of hair follicle epithelium and epidermal keratinocytes, but is common in
stem cells for other cell populations. In any case, regulation of growth and differentiation via integrins, cadherins, and catenins is thought to be working in hair follicle epithelium as well as in epidermal keratinocytes, and from our results cell adhesion molecules, including integrins, cadherins, and catenins, are suggested to be key regulators of growth and differentiation of stem cells for both hair follicle epithelium and epidermal keratinocytes.
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We thank Dr. Margaret J. Wheelock, Department of Biology, University of Toledo, Toledo, OH, U.S.A., for providing us the antibodies, Jelly, 5H10 and 4F11; Dr. Irene M. Leigh, Imperial Cancer Research Fund, London, UK, for a kind gift of the antibody, LP2K; Ms. Megumi Sato, Ms. Yuriko Kanzaki, Ms. Yu Umebayashi, Ms. Marcia L. Usui, and Mr Robert A. Underwood for their ®ne technical assistance on this project. This work was supported in part by Grant-in-Aid for Encouragement of Young Scientists (No. 08770700 and no. 9770668) to M.A. from the Ministry of Education, Science, Sports and Culture of Japan; a Grant-in-Aid from the Cosmetology Research Foundation, Japan to M.A.; grants to L.T.S. (HD-17664 and AR-21557) from the National Institutes of Health, U.S.A; and support from the George F. Odland Endowment Funds.
REFERENCES Adams JC, Watt FM: Regulation of development and differentiation by the extracellular matrix. Development 117:1183±1198, 1993 Akiyama M, Dale BA, Sun T-T, Holbrook KA: Characterization of hair follicle bulge in human fetal skin; the human fetal bulge is a pool of undifferentiated keratinocytes. J Invest Dermatol 105:844±850, 1995 Akiyama M, Smith LT, Holbrook KA: Growth factor and growth factor receptor localization in the hair follicle bulge and associated tissue in human fetus. J Invest Dermatol 106:391±396, 1996 Albelda SM, Buck CA: Integrins and other cell adhesion molecules. FASEB J 4:2868±2880, 1990 Barth AIM, NaÈthke IS, Nelson WJ: Cadherins, catenins and APC protein: interplay between cytoskeletal complexes and signaling pathways. Curr Opin Cell Biol 9:683±690, 1997 Bergelson JM, Shepley MP, Chan BMC, Hemler ME, Finberg RW: Identi®cation of the integrin VLA-2 as a receptor for echo virus 1. Science 255:1718±1720, 1992 Braga VMM, Hodivala KJ, Watt FM: Calcium-induced changes in distribution and solubility of cadherins, integrins and their associated cytoplasmic proteins in human keratinocytes. Cell Adhesion Commun 3:201±215, 1995 Carroll JM, Romero MR, Watt FM: Suprabasal integrin expression in the epidermis of transgenic mice results in developmental defects and a phenotype resembling psoriasis. Cell 83:957±968, 1995 Carter WG, Wayner EA, Bouchard TS, Kaur P: The role of integrins a2b1 and a3b1 in cell-cell and cell-substrate adhesion of human epidermal cells. J Cell Biol 110:1387±1404, 1990 Cerri A, Favre A, Giunta M, Corte G, Grossi CE, Berti E: Immunohistochemical localization of a novel b1 integrin in normal and pathologic squamous epithelia. J Invest Dermatol 102:247±252, 1994 Commo S, Bernard BA: The distribution of a2b1, a3b1 and a6b4 integrins identi®es distinct subpopulations of basal keratinocytes in the outer root sheath of the human anagen hair follicle. Cell Mol Life Sci 53:466±471, 1997 Cotsarelis G, Sun T-T, Lavker RM: 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, 1990 Crossin KL: Cell adhesion molecules in embryogenesis and disease. Ann NY Acad Sci 189:172±186, 1990 Damsky CH, Werb Z: Signal transduction by integrin receptors for extracellular matrix: cooperative processing of extracellular information. Curr Opin Cell Biol 4:772±781, 1992 Damsky CH, Richa J, Solter D, Knudsen K, Buck CA: Identi®cation and puri®cation of a cell surface glycoprotein mediating intercellular adhesion in embryonic and adult tissue. Cell 34:455±466, 1983 De Luca M, Tamura RN, Kajiji S, et al: Polarized integrin mediates human keratinocyte adhesion to basal lamina. Proc Natl Acad Sci USA 87:6888±6892, 1990 Edelman GM, Crossin KL: Cell adhesion molecules: implications for a molecular histology. Annu Rev Biochem 60:155±190, 1991 Hemler ME: VLA proteins in the integrin family: structures, functions, and their role on leukocytes. Annu Rev Immunol 8:365±400, 1990 Hertle MD, Adams JC, Watt FM: Integrin expression during human epidermal development in vivo and in vitro. Development 112:193±206, 1991 Hodivala KJ, Watt FM: Evidence that cadherins play a role in the downregulation of integrin expression that occurs during keratinocyte terminal differentiation. J Cell Biol 124:589±600, 1994 Holbrook KA: Human epidermal embryogenesis. Int J Dermatol 18:329±356, 1979 Holbrook KA, Odland GF: Structure of the human fetal hair canal and initial hair eruption. J Invest Dermatol 71:385±390, 1978 Holbrook KA, Smith LT, Kaplan ED, Minami SA, Hebert GP, Underwood RA: Expression of morphogens during human follicle development in vivo and a model for studying follicle morphogenesis in vitro. J Invest Dermatol 101:39S± 49S, 1993 Hynes RO: Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69:11±25, 1992 Jones PH, Watt FM: Separation of human epidermal stem cells from transit
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amplifying cells on the basis of differences in integrin function and expression. Cell 73:713±724, 1993 Jones PH, Harper S, Watt FM: Stem cell patterning and fate in human epidermis. Cell 80:83±93, 1995 Kaufmann R, FroÈsch D, Westphal C, Weber L, Klein CE: Integrin VLA-3: ultrastructural localization at cell-cell contact sites of human cell cultures. J Cell Biol 109:1807±1815, 1989 Lane EB, BaÂrtek J, Purkis PE, Leigh IM: Keratin antigens in differentiating skin. Ann NY Acad Sci 455:241±258, 1985 Lane EB, Wilson CA, Hughes BR, Leigh IM: Stem cells in hair follicles: cytoskeletal studies. Ann NY Acad Sci 642:197±213, 1991 Larjava H, Peltonen J, Akiyama SK, Yamada SS, Gralnick HR, Uitto J, Yamada KM: Novel function for b1 integrins in keratinocyte cell±cell interactions. J Cell Biol 110:803±815, 1990 Lavker RM, Cotsarelis G, Wei ZG, Sun T-T: Stem cells of pelage, vibrissae and eyelash follicles: the hair cycle and tumor formation. Ann NY Acad Sci 642:214±225, 1991 Lavker RM, Miller S, Wilson C, Cotsarelis G, Wei Z-G, Yang J-S, Sun T-T: Hair follicle stem cells: their location, role in hair cycle, and involvement in skin tumor formation. J Invest Dermatol 101:16S±26S, 1993 Lyle S, Christo®dou-Solomidou M, Liu Y, Elder DE, Albelda S, Cotsarelis G: The C8/144B monoclonal antibody recognizes cytokeratin 15 and de®nes the location of human hair follicle stem cells. J Cell Sci 111:3179±3188, 1998 Madsen A: Studies on the ``bulge'' (Wulst) in super®cial basal cell epithelioma. Arch Dermatol 89:698±708, 1964 Marchisio PC, Bondanza S, Cremona O, De Cancedda R, Luca M: Polarized expression of integrin (a6b4, a2b1, a3b1, and avb5) and their relationship with the cytoskeleton and basement membrane matrix in cultured human keratinocytes. J Cell Biol 112:761±773, 1991 Mercer BM, Sklar S, Shariatmadar A, Gillieson MS, D'Alton ME: Fetal foot length as a predictor of gestational age. J Obstet Gynecol 156:350±355, 1987 Michel M, Torok N, Godbout MJ, Lussier M, Gaudreau P, Royal A, Germain L: Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage. J Cell Sci 109:1017±1028, 1996 Miyamoto S, Teramoto H, Coso OA, Gutkind JS, Burbelo PD, Akiyama SK, Yamada KM: Integrin function: molecular hierarchies of cytoskeletal and signaling molecules. J Cell Biol 131:791±805, 1995 MoleÁs JP, Watt FM: The epidermal stem cell compartment: variation in expression levels of E-cadherin and catenins within the basal layer of human epidermis. J Histochem Cytochem 45:867±874, 1997 Moll I: Proliferative potential of different keratinocytes of plucked human hair follicles. J Invest Dermatol 105:14±21, 1995 Perry TB, Holbrook KA, Hoff MS, Hamilton EF, Senikas V, Fisher C: Prenatal diagnosis of congenital nonbullous ichthyosiform erythroderma (lamellar ichthyosis). Prenat Diagn 7:145±155, 1987 Reynolds ES: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208±212, 1963 Reynolds AJ, Lawrence CM, Jahoda CA: Human hair follicle germinative epidermal cell culture. J Invest Dermatol 101:634±638, 1993 Richardson KC, Jarett L, Finke EH: Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol 35:313±323, 1960 Rochat A, Kobayashi K, Barrandon Y: Location of stem cells of human hair follicles by clonal analysis. Cell 76:1063±1073, 1994 Shepard TH: Normal and abnormal growth patterns. Growth and development of the human embryo and fetus. In: Gardner LI (eds). Endocrine and Genetic Diseases of Childhood and Adolescence. Philadelphia: W.B. Saunders, 1975, pp. 1±8 Shimoyama Y, Hirohashi S, Hirano S, Noguchi M, Shimosato Y, Takeichi M, Abe O: Cadherin cell-adhesion molecules in human epithelial tissues and carcinomas. Cancer Res 49:2128±2133, 1989 Springer TA: Adhesion receptors of the immune system. Nature 346:425±434, 1990 Sun T-T, Cotsarelis G, Lavker RM: Hair follicle stem cells: the bulge-activation hypothesis. J Invest Dermatol 96:77S±78S, 1991 Symington BE, Takada Y, Carter WG: Interactions of integrins a2b1 and a3b1: potential role in keratinocyte intercellular adhesion. J Cell Biol 120:523±535, 1993 Takeichi M: Morphogenetic roles of classic cadherins. Curr Opin Cell Biol 7:619±627, 1995 Watt FM: Epidermal stem cells: markers, patterning and the control of stem cell fate. Phil Trans R Soc Lond B 353:831±837, 1998 Wayner EA, Carter WG: Identi®cation of multiple cell adhesion receptors for collagen and ®bronectin in human ®brosarcoma cells possessing unique a and common b subunits. J Cell Biol 105:1873±1884, 1987 Werb Z, Tremble PM, Behrendtsen O, Crowley E, Damsky CH: Signal transduction through the ®bronectin receptor induces collagenase and stromelysin gene expression. J Cell Biol 109:877±889, 1989 Yang J-S, Lavker RM, Sun T-T: Upper human hair follicle contains a subpopulation of keratinocytes with superior in vitro proliferative potential. J Invest Dermatol 101:652±659, 1993 Zhu AJ, Watt FM: Expression of a dominant negative cadherin mutant inhibits proliferation and stimulates terminal differentiation of human epidermal keratinocytes. J Cell Sci 109:3013±3023, 1996
*Department of Dermatology, Teikyo University School of Medicine, Ichihara Hospital, Chiba, Japan; ²Division of Dermatology, Kitasato Institute Hospital, Tokyo, Japan; ³Departments of Biological Structure and Medicine (Dermatology), University of Washington School of Medicine, Seattle, Washington, U.S.A.; §Department of Dermatology, Hokkaido University School of Medicine, Sapporo, Japan; ¶Department of Dermatology, Keio University School of Medicine, Tokyo, Japan
In rodents, the hair follicle stem cells lie in a wellde®ned bulge in the outer root sheath; however, the bulge as a stem cell site of human hair follicle epithelium is still controversial. Epidermal stem cells are thought to express high levels of b1 integrin and low levels of E-cadherin and b- and g-catenin. In order to clarify the ontogenic distribution of possible stem cells during hair follicle development, the expression patterns of b1 integrin subunits, E-cadherin, and band g-catenins in the skin samples from human fetuses of a series of estimated gestational ages (EGA) were examined. b1 integrin-rich, E-cadherin-, and b- and g-catenin-poor cells, possible stem cells, were localized to the entire hair germ (65±84 d EGA) and later to the outermost cells of hair peg (85±104 d EGA). In the bulbous hair peg (105±135 d EGA) and in the differentiated lanugo hair follicle (>135 d
EGA), they were settled in the bulge and the outermost layer of the outer root sheath. This sequential localization was similar to that of cells rich in epidermal growth factor receptor expression and positive with keratin 19, a putative marker of epidermal stem cells. In addition, these b1 integrin-rich, E-cadherin-, and b- and g-catenin-poor cells showed similar, undifferentiated morphologic features by electron microscopy. This information of ontogenic localization of possible hair follicle stem cells contributes to the further understanding of mechanisms of human hair follicle morphogenesis and supports the idea that the human fetal hair follicle bulge is a site of stem cells for follicular epithelium. Key words: cell adhesion molecule/electron microscopy/epidermal growth factor receptor/fetal development/keratin 19. J Invest Dermatol 114:321±327, 2000
T
was mostly marked in the intermediate part and the lower half of the central part of plucked human hair follicles. Thus, there is a discrepancy between in vivo and in vitro ®ndings and the localization sites of stem cells of human developing hair follicle are still controversial. Integrins, surface receptors mediating cell±cell and cell±extracellular matrix adhesion relevant for a number of physiologic and pathologic phenomena (Albelda and Buck, 1990; Crossin, 1990; Hemler, 1990; Springer, 1990; Edelman and Crossin, 1991), regulate cell adhesion, shape, motility, intracellular signaling, and gene expression (Werb et al, 1989; Springer, 1990; Hynes, 1992). The b1 family of integrins, including a2b1 and a3b1 integrins on epithelial cells, are believed to function in cell±cell interaction, cellsubstrate adhesion (Symington et al, 1993; Larjava et al, 1990; Marchisio et al, 1991), signal transduction for extracellular matrix (Damsky and Werb, 1992; Miyamoto et al, 1995), cell±cell/cell± substrate adhesion (Carter et al, 1990; Marchisio et al, 1991), and cell fate (Jones and Watt, 1993; Symington et al, 1993) of human keratinocytes. Recently, experiments with cultured keratinocytes have established that b1 integrins, including a2b1 and a3b1 integrins, not only mediate cell adhesion and migration, but also regulate strati®cation and the initiation of epithelial differentiation and morphogenesis (Hertle et al, 1991; Adams and Watt, 1993). Furthermore, stem cells are thought to express higher levels of the a2b1 and a3b1 integrins in human epidermis than keratinocytes of lower proliferative potential (Jones and Watt, 1993; Jones et al, 1995; MoleÁs and Watt, 1997).
he hair follicle stem cells have been reported to lie in the bulge region of hair follicles in rodents (Cotsarelis et al, 1990). The human hair follicle bulge is also hypothesized as a stem cell site (Lavker et al, 1991, 1993; Sun et al, 1991). The ``bulge-activation hypothesis'', proposing that hair cycles re¯ected activation and inactivation of stem cells in the bulge, has been developed on the basis of the identi®cation of putative hair follicle stem cells in the bulge region (Cotsarelis et al, 1990; Sun et al, 1991). Recently, b1 integrin bright cells were identi®ed in adult human hair follicle bulges, as was keratin 15 (Lyle et al, 1998). In addition, label-retaining cells were found in the human hair follicle bulge and the human adult hair follicle bulge was strongly suggested to be the stem cell site for hair follicle epithelium (Lyle et al, 1998). From the results of in vitro studies, however, hair follicle stem cells are reported to be either close to the attachment site of the arrector pili muscle (Yang et al, 1993) or slightly lower down (Rochat et al, 1994), or in the follicular bulb (Reynolds et al, 1993) in adult human hair follicles. Moll (1995) reported that colony-forming ability
Manuscript received February 16, 1999; revised October 29, 1999; accepted for publication November 3, 1999. Reprint requests to: Dr. Masashi Akiyama, Department of Dermatology, Teikyo University School of Medicine, Ichihara Hospital, 3426±3, Anesaki, Ichihara, Chiba 299±0111, Japan. Email: [email protected] Abbreviations: EGA, estimated gestational age; K19, keratin 19. 0022-202X/00/$ 15.00
´ Copyright # 2000 by The Society for Investigative Dermatology, Inc. 321
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Cadherins were reported to play a major role in epithelial morphogenesis (Takeichi, 1995) and may regulate keratinocyte growth and differentiation (Zhu and Watt, 1996). Cadherinmediated adhesion is involved in the downregulation of integrin expression that occurs during keratinocyte terminal differentiation (Hodivala and Watt, 1994). The mechanism by which cadherins could regulate integrin expression is presently under investigation and is believed to involve the mutual association of integrins and cadherins with the actin cytoskeleton, which, in the case of cadherins, is mediated by catenins (Braga et al, 1995). There is clear evidence that b-catenin has signaling functions that may be independent of its role in cell±cell adhesion (Barth et al, 1997). In fact, inversely correlated expression of levels of b1 integrins and E-cadherin or b-catenin in basal keratinocytes of the interfollicular epidermis were reported (MoleÁs and Watt, 1997). Thus, dull stainings for E-cadherin and b-catenin are thought to be a stem cell marker (Watt, 1998). Ontogenically, human hair follicle development starts as a hair germ [65±84 d estimated gestational age (EGA)], which is a bud from the epidermis into the dermis and is associated with an underlying condensation of mesenchymal cells (Holbrook and Odland, 1978; Holbrook, 1979; Holbrook et al, 1993). In the next stage of development, the hair peg (85±104 d EGA) forms as the cord of epidermal cells growing into the dermis. In the hair follicle of bulbous hair peg stage (105±135 d EGA), each part of the follicle begins to differentiate into regions that are de®ned by the position of outgrowths of outer root sheath cells including the sebaceous gland primordium and the bulge. The ¯at end of the hair peg molds into a bulb and the associated mesenchymal cells become the dermal papilla. Finally, differentiated hair follicles that generate the thin hair seen at birth named ``lanugo hair'' are formed in the late second trimester (>135 d EGA). In this study, we investigated the expression of b1, a2, and a3 integrins, E-cadherin, and b- and g-catenins in the developing human hair follicles of skin samples from human fetuses of a series of EGA (49±163 d EGA) in order to elucidate whether these cell adhesion molecules are involved in human hair follicle morphogenesis, and to clarify the stem cell sites in the early stage of developing human fetal hair follicles. In addition, we identi®ed b1 integrin-rich, E-cadherin-, and b- and g-catenin-poor cells and compared these cells with keratin 19 (K19)-positive, epidermal growth factor (EGF) receptor-rich cells, based on the evidence that K19 is a marker for epidermal stem cells (Lane et al, 1991; Michel et al, 1996) and that the stem cells strongly express EGF receptor (Akiyama et al, 1996). We also observed the ultrastructural features of these possible stem cells (b1 integrin-rich, E-cadherin-, and band g-catenin-poor) in human developing hair follicles. Our ®ndings demonstrated that possible stem cells for hair follicle epithelium were present in the entire hair germ, became localized ®rst to outermost cells of hair peg, and then in the bulge and the outermost layer of the outer root sheath (ORS). This sequential localization was similar to that of K19-positive, EGF receptor-rich cells. Ultrastructurally, these cells at any stage of hair follicle development showed similar, undifferentiated features that distinguished them from other follicular cells. These ®ndings of unique sequential localization of potential follicular stem cells are important for understanding the mechanisms of initiation, induction, and development of human fetal hair follicles. MATERIALS AND METHODS Tissue Human embryonic and fetal skin specimens were obtained from abortuses of 49±163 d EGA through the Central Laboratory of Human Embryology at the University of Washington, Seattle, U.S.A. with the approval of the Human Subjects Review Board and in accordance with the United States DHEW policies. The ages, the autopsy sites, and the numbers of embryos or fetuses included in this study were as follows: 49±64 d EGA, scalp (n = 2), trunk (n = 2); 65±84 d EGA, scalp (n = 3), trunk (n = 2); 85± 104 d EGA, scalp (n = 2), trunk (n = 2); 105±135 d EGA, scalp (n = 2), trunk (n = 2); >135 d EGA, scalp (n = 2), trunk (n = 2). Two or three skin specimens from each fetus were used for the study. EGA was determined
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from maternal histories, fetal measurements (crown rump and foot length), and comparative histologic appearance of epidermis (Shepard, 1975; Holbrook, 1979; Mercer et al, 1987). Antibodies The primary antibodies used in this study were mouse monoclonal antihuman a2 integrin antibody, Clone P1E6 (Carter et al, 1990) (DAKO, Carpinteria, CA), mouse monoclonal antihuman a3 integrin antibody, Clone P1B5 (Wayner and Carter, 1987) (DAKO), mouse monoclonal antihuman b1 integrin antibody, Clone DE9 (Bergelson et al, 1992) (Upstate Biotechnology, Lake Placid, NY), mouse monoclonal antihuman E-cadherin antibody, HECD-1 (Shimoyama et al, 1989), rabbit polyclonal antihuman E-cadherin antibody, Jelly (Damsky et al, 1983), mouse monoclonal anti-b catenin antibodies, Clone 14 (Transduction Laboratories, Lexington, KY) and 5H10, mouse monoclonal anti-g catenin antibody, 4F11, mouse monoclonal antihuman EGF receptor antibody, Clone EGFR1 (DAKO, Glostrup, Denmark), and mouse monoclonal antihuman K19 antibody, LP2K (Lane et al, 1985). Immuno¯uorescent labeling Fetal skin was quick frozen in a dry iceethanol slush and 6 mm thick sections were cut using a cryostat. At least three sections from each specimen were stained and examined. The sections were incubated in normal goat serum for 30 min and then incubated in primary antibody solution for 1 h at 37°C. Antibody dilutions were 1:50 for anti-a2 integrin antibody and anti-a3 integrin antibody, 1:40 for anti-b1 integrin antibody, 1:500 for HECD-1, 1:160 for Jelly and antib-catenin, clone 14, 1:5 for anti-EGF receptor antibody, and 1:10 for LP2K. Antibodies 5H10 and 4F11, were used neat. The sections were then incubated in ¯uorescein isothiocyanate (FITC)-conjugated to rabbit antimouse immunoglobulins (DAKO) or, for polyclonal antibody, Jelly, incubated in FITC-conjugated to goat antirabbit immunoglobulins (DAKO) for 30 min at room temperature, followed by 10 mg per ml propidium iodide to counterstain nuclei (Sigma, St. Louis, MO) for 10 s. The sections were extensively washed with phosphate-buffered saline between incubations. The stained sections were mounted with a cover slip in 50% glycerol mounting medium and stored in the refrigerator. Photos were taken with epi¯uorescent microscopy for several days after the immunostaining. Speci®c immunostainings were detected as green (FITC) and nuclear stain was observed as red (propidium iodide). Overlap of both FITC and propidium iodide was demonstrated as a yellowish color. Image analysis Image analysis for photo slides of a conventional immuno¯uorescence microscope was done using a image modi®cation software, Adobe Photoshop 3.0 (Adobe Systems, Mountain View, CA). In order to highlight the brightly stained cells, the strong signals of FITC (stronger than 20% of only green signals in histograms of distribution of signal level of pixels) were picked up from the images using a command, input-level adjustment. In order to reduce the contamination of propidium iodide ¯uorescence, all the red signals were weakened equally before the image analysis. A set exposure time was used for all the photo slides for image analysis. Electron microscopy Fetal skin was ®xed in one-half strength Karnovsky's ®xative or 2% glutaraldehyde solution, post®xed in 1% OsO4, dehydrated, and embedded in Epon 812 (Perry et al, 1987). All the samples were serially sectioned, sampled every 10±15 mm for light microscopy (1 mm thick), and thin sectioned for electron microscopy (70 nm thick). The histologic sections were stained by the method of Richardson et al (1960). The thin sections were stained with uranyl acetate and lead citrate (Reynolds, 1963) and examined under a JEOL 1200EXII transmission electron microscope in the transmission mode at 80 kV.
RESULTS b1 integrin-rich, E-cadherin-, and b- and g-catenin-poor cells were localized to the entire hair germ (65±84 d EGA) and the outer cells of the hair peg (85±104 d EGA) in the early development As for the results of all immunostainings, a consistent pattern was obtained in all the sections stained with each antibody at each developmental stage. Strong membranous immunostainings of b1, a2, and a3 integrin subunits were observed in the entire hair germ (Fig 1). At the same time, these b1 integrin-rich cells were E-cadherin- and b- and g-catenin-poor compared with suprabasal keratinocytes of the interfollicular epidermis. The outermost cells of hair peg showed strong membranous stainings of b1, a2, and a3 integrins (Fig 1). The outermost cells of hair peg were E-cadherin- and b- and g-catenin-
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Figure 1. b1 integrin-rich cells are seen in the entire hair germ, in the outer layer of the hair peg, and in the outermost layer of the early bulbous hair peg. Hair germ cells express g-catenin only weakly compared with those of interfollicular epidermis (a, b). b1 integrin is strongly expressed in the entire hair germ (c) and the outer cells of the hair peg (e). The expression of g-catenin is weak in the outer cells of the hair peg (d). a2 integrin is brightly expressed in the outermost cells of the early bulbous hair peg and the basal layer of the interfollicular epidermis (f). Speci®c immunostainings are demonstrated with ¯uorescein isothiocyanate (FITC, green) and nuclear stain is done with propidium iodide (red). Yellowish stain is caused mainly by the overlap of FITC ¯uorescence and propidium iodide ¯uorescence and partly by the nonspeci®c detection of FITC ¯uorescence by the ®lter for propidium iodide. (a±c) Hair germ (65±84 d EGA), (d, e) hair peg (85±104 d EGA), (f) early bulbous hair peg (108 d EGA), (c, e) b1 integrin, (f) a2 integrin, (a, b, d) g-catenin (4F11); scale bars: 50 mm.
poor compared with the inner cells of hair peg. The inner cells of hair peg were b1, a2, and a3 integrin-poor and E-cadherin- and b- and g-catenin-rich compared with the outermost cells. b1 integrin-rich, E-cadherin-, and b- and g-catenin-poor cells were speci®cally localized to the bulge and the outermost layer of ORS in the bulbous hair peg (105±135 d EGA) and the lanugo hair follicle (>135 d EGA) In the early bulbous hair peg, b1, a2, and a3 integrin-rich cells were localized to the outer cells all around the peg (Fig 1). These b1, a2, and a3 integrin-rich cells were E-cadherin-, b-, and g-catenin-dull compared with the inner cells (Fig 1). In the bulbous hair peg, bright membranous stainings of b1, a2, and a3 integrins were observed in the bulge and the outer cells of ORS. These b1 integrin-rich cells in the bulge and the outer cells of ORS were Ecadherin-, b-, and g-catenin-poor. In the differentiated lanugo hair follicle, bright staining of b1, a2, and a3 integrins was observed in the outer cells of the ORS and was weak in the inner cells of the ORS (Fig 2). a2 subunit is also brightly expressed in the matrix cells of the bulb. b1 and a3 integrins were only weakly expressed in the matrix cells of the bulb. In addition, the matrix cells of the bulb were strongly stained with b- and g-catenins, although the matrix cells were E-cadherin-poor. Thus, the b1 integrin-rich, E-cadherin-, b-, and g-catenin-poor cells were localized to the bulge and the outermost layer of the ORS both in the bulbous hair peg stage and in the lanugo hair follicle stage. Image modi®cation con®rmed the localization of b1 integrin-rich, E-cadherin-, b-, and g-catenin-poor cells in the bulge and the outermost layer of the ORS By the deletion of weak signals using image modi®cation, only the bulge cells and the outer layer cells of the ORS were revealed to exhibit
remaining strong signals for b1, a2, and a3 integrins (Fig 3). Image analysis revealed that the bulge region showed weak signals for Ecadherin and b- and g-catenins. These b1, a2, and a3 integrin-rich cells were E-cadherin-, b-, and g-catenin-poor. K19-positive cells and EGF receptor-rich cells showed similar ontogenic localization patterns to that of b1 integrin-rich, E-cadherin-, b-, and g-catenin-poor cells K19-positive cells were sequentially restricted to the hair germ, to the outer layer of hair peg, to the bulge and the outermost layer of the ORS in the bulbous hair peg and the lanugo hair follicles (Fig 4a). Cells in the hair germ and the outer layer of hair peg expressed EGF receptor strongly. Later, intense immunoreactivity of EGF receptor was seen in the cells in the bulge and the outermost layer of the ORS in the bulbous hair peg and the lanugo hair follicles (Fig 4b). The cells in the sites of ontogenic localization of b1 integrinrich, E-cadherin-, b-, and g-catenin-poor cells showed uniformly undifferentiated morphology Ultrastructurally, hair germ cells and cells in the outer layer of hair peg exhibited similar, undifferentiated features, including abundance of free ribosomes and glycogen particles in the cytoplasm and reduced number of cytoplasmic organelles indicative of differentiation, e.g., rough endoplasmic reticulum and intermediate ®laments (Fig 5a, b). In the bulbous hair peg and the lanugo hair follicle, bulge cells and cells in the outermost layer of the ORS showed similar, undifferentiated features to those seen in the cells in hair germ and the outer layer of the hair peg ultrastructurally (Fig 5c, d). Especially, the bulge consists of uniform, small undifferentiated cells.
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Figure 2. b1 integrin-rich, E-cadherin-, b-catenin-, g-catenin-poor cells are seen in the outermost layer of the ORS, but not in the matrix of the bulb in the lower portion of differentiated lanugo hair follicle (>135 d EGA). Bright stainings of b1 integrin (a), a2 integrin (b), and a3 integrin (c) are observed in the outermost layer (arrowheads) of the ORS. The outermost layer (arrowheads) of the ORS expresses Ecadherin (d), b-catenin (e), and g-catenin (f) very weakly. The matrix cells of the bulb are strongly stained with a2 integrin (b) and weakly stained with E-cadherin (d); however, in contrast to the outermost layer of the ORS, the matrix cells of the bulb express b1 integrin (a) and a3 integrin (c) only weakly and are brightly stained with b-catenin (e) and g-catenin (f). Speci®c immunostainings are demonstrated with FITC (green) and nuclear stain is done with propidium iodide (red). (a) b1 integrin, (b) a2 integrin, (c) a3 integrin, (d) E-cadherin (HECD-1), (e) b-catenin (Clone 14), (f) g-catenin (4F11); scale bar: 50 mm.
Possible stem cells were localized to the entire hair germ, and later to the outer cells of the hair peg, and were settled in the outermost layer of the ORS and the bulge of the bulbous hair peg and the lanugo hair follicle Summarizing the data shown above, possible stem cells, which were b1 integrinrich, E-cadherin- and b-catenin-poor, K19-positive and EGF receptor-rich, and undifferentiated in morphology, were sequentially localized to the entire hair germ and to the outermost cells of hair peg. Later in the bulbous hair peg stage and in the lanugo hair follicle stage, these possible stem cells were seen restrictively in the bulge and the outermost layer of the ORS (Fig 6). DISCUSSION In this study, unique sequential expression of b1, a2, and a3 integrin subunits, E-cadherin, and b- and g-catenins were shown at each stage of human hair follicle development. According to the results, strong stainings of b1, a2, and a3 integrins were seen in the entire hair germ (65±84 d EGA) and in the outermost cells of hair peg (85±104 d EGA) of the early stage of human hair follicle development. These results imply that cell±cell interaction and cell±extracellular matrix adhesion mediated by a2b1 and a3b1 integrins may be involved in early induction of human hair follicle morphogenesis. Based on the plural evidence that the b1 integrin-rich, Ecadherin-and b- and g-catenin-poor cells are stem cells of keratinocytes (MoleÁs and Watt, 1997), these results indicated that the entire hair germ consists of possible pluripotent stem cells and, in the next stage, that the possible stem cells are restricted only to the outer layer in the hair peg. Finally in the late stage of follicular development, possible stem cells of the hair follicle epithelium are thought to be localized in the bulge and the outermost layer of the
ORS (Fig 6). Our ®ndings of the dynamic ontogenic localization of possible stem cells provide important clues to understanding the mechanism of human hair follicle development. In developing hair follicles in human fetuses, the bulge is a prominent hemispherical protrusion (Madsen, 1964) consisting of undifferentiated cells that are immunolabeled with an antibody to K19 (Lane et al, 1985; Akiyama et al, 1995), a possible stem cell marker (Lane et al, 1991; Michel et al, 1996). Keratin 15-positive, b1 integrin bright cells were identi®ed in adult human hair follicle bulges (Lyle et al, 1998). In addition, label-retaining cells were found in the adult human hair follicle bulge (Lyle et al, 1998). The human fetal bulge cells are also known to strongly express EGF receptor (Akiyama et al, 1996). These observations were con®rmed in this study. The existence of K19 keratin and strong expression of EGF receptor in the bulge cells may support the notion that the bulge is a hair follicle stem cell site also in human. Our observations show that cells in the bulge are unique in that they are uniformly b1 integrin-rich, E-cadherin-, and b- and g-catenin-poor. These facts further support the notion that the bulge is a stem cell site of hair follicle epithelium at least during the fetal period. In the epidermis of human skin, expression of the b1 integrin in vivo and in culture is con®ned to the basal cells in contact with basement membrane (Kaufman et al, 1989; De Luca et al, 1990; Hertle et al, 1991; Cerri et al, 1994). In adult human anagen hair follicles, a shift in expression of a2b1 and a3b1 integrins by the outer ORS cells from a basolateral distribution (basement membrane zone) in the lower ORS to an apicolateral expression (nonbasal side) in the upper ORS was reported (Commo and Bernard, 1997). These altered localization patterns may re¯ect the two different functions of a2b1 and a3b1 integrins, i.e., cell±cell adhesion and cell±extracellular matrix interaction. Interestingly, in this study, all cells in the fetal bulge including the inner cells, which are not in contact with the extracellular matrix, showed strong
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Figure 3. The modi®ed images clearly demonstrate that the bulge of the lanugo hair follicle (>135 d EGA) consists of b1 integrinrich, E-cadherin-, b-catenin-, g-catenin-poor cells. Deletion of the weak signals from the immuno¯uorescent images of the mid-portion of the lanugo hair follicle revealed that the strong signals of b1 integrin (a), a2 integrin (b), and a3 integrin (c) remain in the bulge (arrowheads), although cells in the bulge (arrowheads) show no strong signals of E-cadherin (d), b-catenin (e), and g-catenin (f). Speci®c immunostainings are demonstrated with FITC (green) and nuclear stain is done with propidium iodide (red). (a) b1 integrin, (b) a2 integrin, (c) a3 integrin, (d) E-cadherin (HECD-1), (e) b-catenin (5H10), (f) g-catenin (4F11); scale bar: 50 mm.
stainings for b1, a2, and a3 integrin subunits. In addition, the fetal bulge cells exhibited poor expression of E-cadherin and b- and gcatenins. These facts suggest that the fetal bulge cells are a uniform and unique population in terms of expression of cell adhesion molecules on the cell surface. Carroll et al (1995) established b1 integrin transgenic mice using the involucrin promoter that express functional human b1 integrin in the suprabasal epidermal layers. The b1 integrin transgenic mice showed abnormalities of the hairs of the coat and whiskers. The abnormal coat phenotype was apparent as soon as the ®rst coat developed, and there was a progressive normalization through successive hair growth cycles. These ®ndings suggest that regulated b1 integrin expression is essential for the hair follicle development, although the exact mechanisms of functions of b1 integrins for the hair follicle morphogenesis are still unknown. In this context, our ®ndings of ontogenic localization of b1 integrin expression is important to elucidate the roles of b1 integrins in human hair follicle development. In this study, the selected criteria for follicle stem cell stainings, b1 integrins high, E-cadherin-low, b- and g-catenins-low, were extrapolated from work with epidermal stem cell (MoleÁs and Watt, 1997). Our results not only demonstrated the patterns of localization of putative stem cells in human developing hair follicle, but also revealed that the stainings for E-cadherin, b- and g-catenins are useful markers for stem cells of follicular epithelium as well as epidermal stem cells, at least in the fetal period. Existence of a population of skin pluripotent stem cells has been hypothesized (Cotsarelis et al, 1990). In the hypothesis, the pluripotent stem cells are thought to provide transient amplifying cells not only to hair follicles, but also to the epidermis. These
Figure 4. K19-positive, EGF receptor-rich cells are also seen in the bulge and the outermost layer of the lanugo hair follicle. (a) In the lanugo hair follicle (136 d EGA), cells in the bulge (arrows) and in the outermost layer of the ORS (arrowheads) are K19-positive. (b) Cells in the bulge (arrows) and in the outermost layer of the ORS (arrowheads) show stronger immunoreactivity for EGF receptor than the other parts of the developing hair follicle. (a) K19 staining; (b) EGF receptor staining; arrows, bulge; arrowheads, outermost layer of ORS; scale bar: 50 mm.
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Figure 5. Cells in the hair germ, the outer layer of hair peg, and the bulge and the outermost layer of the bulbous hair peg show similar, undifferentiated morphologic features. Electron microscopy reveals that hair germ cells (a), cells in the outer layer of hair peg (b), cells in the bulge of bulbous hair peg (c), and cells in the outermost layer of the ORS of bulbous hair peg (d) show a reduced amount of intermediate ®lament bundles and rough endoplasmic reticulum, and abundance of glycogen particles and free ribosomes in the cytoplasm. The nucleus/cytoplasm ratio of these cells is high and chromatin aggregation in the nuclei appears poor. (a) Cells in the hair germ (69 d EGA); (b) cells in the outer layer of hair peg (89 d EGA); (c) cells in the bulge of bulbous hair peg (125 d EGA); (d) cells in the outermost layer of the ORS of bulbous hair peg (125 d EGA); scale bars: 1 mm.
Figure 6. Sequential localization of b1 integrin-rich, E-cadherin-, b-catenin-, g-cateninpoor cells during human fetal hair follicle development. Ontogenically, b1 integrin-rich, Ecadherin-, and b- and g-catenin-poor cells, possible hair follicle stem cells, are restricted to the entire hair germ, the outer cells of hair peg, then the bulge and the outer layer cells of the bulbous hair peg, and ®nally the bulge and the outermost cells of the ORS of lanugo hair follicle. Dotted area, restriction sites of b1 integrin-rich, E-cadherin-, and b- and g-catenin-poor cells; arrows, the bulge.
results demonstrated that stem cells of hair follicle epithelium and those of epidermal keratinocytes have similar features of expression to cell adhesion molecules and this fact may support the idea of the existence of a skin pluripotent stem cell population shared by the hair follicle epithelium and the epidermal keratinocytes. On the other hand, there is a possibility that the characteristic expression pattern of adhesion molecules is not speci®c to stem cells of hair follicle epithelium and epidermal keratinocytes, but is common in
stem cells for other cell populations. In any case, regulation of growth and differentiation via integrins, cadherins, and catenins is thought to be working in hair follicle epithelium as well as in epidermal keratinocytes, and from our results cell adhesion molecules, including integrins, cadherins, and catenins, are suggested to be key regulators of growth and differentiation of stem cells for both hair follicle epithelium and epidermal keratinocytes.
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We thank Dr. Margaret J. Wheelock, Department of Biology, University of Toledo, Toledo, OH, U.S.A., for providing us the antibodies, Jelly, 5H10 and 4F11; Dr. Irene M. Leigh, Imperial Cancer Research Fund, London, UK, for a kind gift of the antibody, LP2K; Ms. Megumi Sato, Ms. Yuriko Kanzaki, Ms. Yu Umebayashi, Ms. Marcia L. Usui, and Mr Robert A. Underwood for their ®ne technical assistance on this project. This work was supported in part by Grant-in-Aid for Encouragement of Young Scientists (No. 08770700 and no. 9770668) to M.A. from the Ministry of Education, Science, Sports and Culture of Japan; a Grant-in-Aid from the Cosmetology Research Foundation, Japan to M.A.; grants to L.T.S. (HD-17664 and AR-21557) from the National Institutes of Health, U.S.A; and support from the George F. Odland Endowment Funds.
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