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Changing Patterns of Localization of Putative Stem Cells in Developing Human Hair Follicles

2000; Elsevier BV; Volume: 114; Issue: 2 Linguagem: Inglês

10.1046/j.1523-1747.2000.00857.x

1523-1747

Masashi Akiyama, Lynne T. Smith, Hiroshi Shimizu,

Dermatologic Treatments and Research

In rodents, the hair follicle stem cells lie in a well-defined 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 β1 integrin and low levels of E-cadherin and β- and γ-catenin. In order to clarify the ontogenic distribution of possible stem cells during hair follicle development, the expression patterns of β1 integrin subunits, E-cadherin, and β- and γ-catenins in the skin samples from human fetuses of a series of estimated gestational ages (EGA) were examined. β1 integrin-rich, E-cadherin-, and β- and γ-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 β1 integrin-rich, E-cadherin-, and β- and γ-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. In rodents, the hair follicle stem cells lie in a well-defined 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 β1 integrin and low levels of E-cadherin and β- and γ-catenin. In order to clarify the ontogenic distribution of possible stem cells during hair follicle development, the expression patterns of β1 integrin subunits, E-cadherin, and β- and γ-catenins in the skin samples from human fetuses of a series of estimated gestational ages (EGA) were examined. β1 integrin-rich, E-cadherin-, and β- and γ-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 β1 integrin-rich, E-cadherin-, and β- and γ-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. estimated gestational age keratin 19 The hair follicle stem cells have been reported to lie in the bulge region of hair follicles in rodents (Cotsarelis et al., 1990Cotsarelis G. Sun T.-T. Lavker R.M. Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis.Cell. 1990; 61: 1329-1337Abstract Full Text PDF PubMed Scopus (1803) Google Scholar). The human hair follicle bulge is also hypothesized as a stem cell site (Lavker et al., 1991Lavker R.M. Cotsarelis G. 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Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis.Cell. 1990; 61: 1329-1337Abstract Full Text PDF PubMed Scopus (1803) Google Scholar;Sun et al., 1991Sun T.-T. Cotsarelis G. Lavker R.M. Hair follicle stem cells: the bulge-activation hypothesis.J Invest Dermatol. 1991; 96: 77S-78SAbstract Full Text PDF PubMed Google Scholar). Recently, β1 integrin bright cells were identified in adult human hair follicle bulges, as was keratin 15 (Lyle et al., 1998Lyle S. Christofidou-Solomidou M. Liu Y. Elder D.E. Albelda S. Cotsarelis G. The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells.J Cell Sci. 1998; 111: 3179-3188PubMed Google Scholar). 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Location of stem cells of human hair follicles by clonal analysis.Cell. 1994; 76: 1063-1073Abstract Full Text PDF PubMed Scopus (447) Google Scholar), or in the follicular bulb (Reynolds et al., 1993Reynolds A.J. Lawrence C.M. Jahoda C.A. Human hair follicle germinative epidermal cell culture.J Invest Dermatol. 1993; 101: 634-638Abstract Full Text PDF PubMed Google Scholar) in adult human hair follicles.Moll, 1995Moll I. Proliferative potential of different keratinocytes of plucked human hair follicles.J Invest Dermatol. 1995; 105: 14-21Crossref PubMed Scopus (63) Google Scholar reported that colony-forming ability 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 findings and the localization sites of stem cells of human developing hair follicle are still controversial. 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Recently, experiments with cultured keratinocytes have established that β1 integrins, including α2β1 and α3β1 integrins, not only mediate cell adhesion and migration, but also regulate stratification and the initiation of epithelial differentiation and morphogenesis (Hertle et al., 1991Hertle M.D. Adams J.C. Watt F.M. Integrin expression during human epidermal development in vivo and in vitro.Development. 1991; 112: 193-206PubMed Google Scholar;Adams and Watt, 1993Adams J.C. Watt F.M. Regulation of development and differentiation by the extracellular matrix.Development. 1993; 117: 1183-1198Crossref PubMed Google Scholar). Furthermore, stem cells are thought to express higher levels of the α2β1 and α3β1 integrins in human epidermis than keratinocytes of lower proliferative potential (Jones and Watt, 1993Jones P.H. Watt F.M. 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Expression of a dominant negative cadherin mutant inhibits proliferation and stimulates terminal differentiation of human epidermal keratinocytes.J Cell Sci. 1996; 109: 3013-3023Crossref PubMed Google Scholar). Cadherin-mediated adhesion is involved in the downregulation of integrin expression that occurs during keratinocyte terminal differentiation (Hodivala and Watt, 1994Hodivala K.J. Watt F.M. Evidence that cadherins play a role in the downregulation of integrin expression that occurs during keratinocyte terminal differentiation.J Cell Biol. 1994; 124: 589-600Crossref PubMed Scopus (202) Google Scholar). 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., 1995Braga V.M.M. Hodivala K.J. Watt F.M. Calcium-induced changes in distribution and solubility of cadherins, integrins and their associated cytoplasmic proteins in human keratinocytes.Cell Adhesion Commun. 1995; 3: 201-215Crossref PubMed Scopus (58) Google Scholar). There is clear evidence that β-catenin has signaling functions that may be independent of its role in cell–cell adhesion (Barth et al., 1997Barth A.I.M. Näthke I.S. Nelson W.J. Cadherins, catenins and APC protein: interplay between cytoskeletal complexes and signaling pathways.Curr Opin Cell Biol. 1997; 9: 683-690Crossref PubMed Scopus (478) Google Scholar). In fact, inversely correlated expression of levels of β1 integrins and E-cadherin or β-catenin in basal keratinocytes of the interfollicular epidermis were reported (Molès and Watt, 1997Molès J.P. Watt F.M. The epidermal stem cell compartment: variation in expression levels of E-cadherin and catenins within the basal layer of human epidermis.J Histochem Cytochem. 1997; 45: 867-874Crossref PubMed Scopus (76) Google Scholar). Thus, dull stainings for E-cadherin and β-catenin are thought to be a stem cell marker (Watt, 1998Watt F.M. Epidermal stem cells: markers, patterning and the control of stem cell fate.Phil Trans R Soc Lond B. 1998; 353: 831-837Crossref PubMed Scopus (318) Google Scholar). 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, 1978Holbrook K.A. Odland G.F. Structure of the human fetal hair canal and initial hair eruption.J Invest Dermatol. 1978; 71: 385-390Crossref PubMed Scopus (28) Google Scholar;Holbrook, 1979Holbrook K.A. Human epidermal embryogenesis.Int J Dermatol. 1979; 18: 329-356PubMed Google Scholar;Holbrook et al., 1993Holbrook K.A. Smith L.T. Kaplan E.D. Minami S.A. Hebert G.P. Underwood R.A. Expression of morphogens during human follicle development in vivo and a model for studying follicle morphogenesis in vitro.J Invest Dermatol. 1993; 101: 39S-49SAbstract Full Text PDF PubMed Scopus (55) Google Scholar). 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 defined by the position of outgrowths of outer root sheath cells including the sebaceous gland primordium and the bulge. The flat 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 β1, α2, and α3 integrins, E-cadherin, and β- and γ-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 identified β1 integrin-rich, E-cadherin-, and β- and γ-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., 1991Lane E.B. Wilson C.A. Hughes B.R. Leigh I.M. Stem cells in hair follicles: cytoskeletal studies.Ann NY Acad Sci. 1991; 642: 197-213Crossref PubMed Scopus (84) Google Scholar;Michel et al., 1996Michel M. Torok N. Godbout M.J. 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. 1996; 109: 1017-1028PubMed Google Scholar) and that the stem cells strongly express EGF receptor (Akiyama et al., 1996Akiyama M. Smith L.T. Holbrook K.A. Growth factor and growth factor receptor localization in the hair follicle bulge and associated tissue in human fetus.J Invest Dermatol. 1996; 106: 391-396Crossref PubMed Scopus (78) Google Scholar). We also observed the ultrastructural features of these possible stem cells (β1 integrin-rich, E-cadherin-, and β- and γ-catenin-poor) in human developing hair follicles. Our findings demonstrated that possible stem cells for hair follicle epithelium were present in the entire hair germ, became localized first 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 findings 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. 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 from maternal histories, fetal measurements (crown rump and foot length), and comparative histologic appearance of epidermis (Shepard, 1975Shepard T.H. Normal and abnormal growth patterns. Growth and development of the human embryo and fetus.in: Gardner L.I. Endocrine and Genetic Diseases of Childhood and Adolescence. W.B. Saunders, Philadelphia1975: 1-8Google Scholar;Holbrook, 1979Holbrook K.A. Human epidermal embryogenesis.Int J Dermatol. 1979; 18: 329-356PubMed Google Scholar;Mercer et al., 1987Mercer B.M. Sklar S. Shariatmadar A. Gillieson M.S. D'Alton M.E. Fetal foot length as a predictor of gestational age.J Obstet Gynecol. 1987; 156: 350-355Abstract Full Text PDF Scopus (110) Google Scholar). The primary antibodies used in this study were mouse monoclonal antihuman α2 integrin antibody, Clone P1E6 (Carter et al., 1990Carter W.G. Wayner E.A. Bouchard T.S. Kaur P. The role of integrins α2β1 and α3β1 in cell-cell and cell-substrate adhesion of human epidermal cells.J Cell Biol. 1990; 110: 1387-1404Crossref PubMed Scopus (506) Google Scholar) (DAKO, Carpinteria, CA), mouse monoclonal antihuman α3 integrin antibody, Clone P1B5 (Wayner and Carter, 1987Wayner E.A. Carter W.G. Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique α and common β subunits.J Cell Biol. 1987; 105: 1873-1884Crossref PubMed Scopus (534) Google Scholar) (DAKO), mouse monoclonal antihuman β1 integrin antibody, Clone DE9 (Bergelson et al., 1992Bergelson J.M. Shepley M.P. Chan B.M.C. Hemler M.E. Finberg R.W. Identification of the integrin VLA-2 as a receptor for echo virus 1.Science. 1992; 255: 1718-1720Crossref PubMed Scopus (244) Google Scholar) (Upstate Biotechnology, Lake Placid, NY), mouse monoclonal antihuman E-cadherin antibody, HECD-1 (Shimoyama et al., 1989Shimoyama 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. 1989; 49: 2128-2133PubMed Google Scholar), rabbit polyclonal antihuman E-cadherin antibody, Jelly (Damsky et al., 1983Damsky C.H. Richa J. Solter D. Knudsen K. Buck C.A. Identification and purification of a cell surface glycoprotein mediating intercellular adhesion in embryonic and adult tissue.Cell. 1983; 34: 455-466Abstract Full Text PDF PubMed Scopus (254) Google Scholar), mouse monoclonal anti-β catenin antibodies, Clone 14 (Transduction Laboratories, Lexington, KY) and 5H10, mouse monoclonal anti-γ catenin antibody, 4F11, mouse monoclonal antihuman EGF receptor antibody, Clone EGFR1 (DAKO, Glostrup, Denmark), and mouse monoclonal antihuman K19 antibody, LP2K (Lane et al., 1985Lane E.B. Bártek J. Purkis P.E. Leigh I.M. Keratin antigens in differentiating skin.Ann NY Acad Sci. 1985; 455: 241-258Crossref PubMed Scopus (276) Google Scholar). Fetal skin was quick frozen in a dry ice-ethanol slush and 6 μm 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-α2 integrin antibody and anti-α3 integrin antibody, 1:40 for anti-β1 integrin antibody, 1:500 for HECD-1, 1:160 for Jelly and anti-β-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 fluorescein 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 μg 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 epifluorescent microscopy for several days after the immunostaining. Specific 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 for photo slides of a conventional immunofluorescence microscope was done using a image modification 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 fluorescence, 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. Fetal skin was fixed in one-half strength Karnovsky's fixative or 2% glutaraldehyde solution, postfixed in 1% OsO4, dehydrated, and embedded in Epon 812 (Perry et al., 1987Perry T.B. Holbrook K.A. Hoff M.S. Hamilton E.F. Senikas V. Fisher C. Prenatal diagnosis of congenital nonbullous ichthyosiform erythroderma (lamellar ichthyosis).Prenat Diagn. 1987; 7: 145-155Crossref PubMed Scopus (20) Google Scholar). All the samples were serially sectioned, sampled every 10–15 μm for light microscopy (1 μm thick), and thin sectioned for electron microscopy (70 nm thick). The histologic sections were stained by the method ofRichardson et al., 1960Richardson K.C. Jarett L. Finke E.H. Embedding in epoxy resins for ultrathin sectioning in electron microscopy.Stain Technol. 1960; 35: 313-323Crossref PubMed Scopus (2398) Google Scholar. The thin sections were stained with uranyl acetate and lead citrate (Reynolds, 1963Reynolds E.S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy.J Cell Biol. 1963; 17: 208-212Crossref PubMed Scopus (17280) Google Scholar) and examined under a JEOL 1200EXII transmission electron microscope in the transmission mode at 80 kV. 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 β1, α2, and α3 integrin subunits were observed in the entire hair germ (Figure 1). At the same time, these β1 integrin-rich cells were E-cadherin- and β- and γ-catenin-poor compared with suprabasal keratinocytes of the interfollicular epidermis. The outermost cells of hair peg showed strong membranous stainings of β1, α2, and α3 integrins (Figure 1). The outermost cells of hair peg were E-cadherin- and β- and γ-catenin-poor compared with the inner cells of hair peg. The inner cells of hair peg were β1, α2, and α3 integrin-poor and E-cadherin- and β- and γ-catenin-rich compared with the outermost cells. In the early bulbous hair peg, β1, α2, and α3 integrin-rich cells were localized to the outer cells all around the peg (Figure 1). These β1, α2, and α3 integrin-rich cells were E-cadherin-, β-, and γ-catenin-dull compared with the inner cells (Figure 1). In the bulbous hair peg, bright membranous stainings of β1, α2, and α3 integrins were observed in the bulge and the outer cells of ORS. These β1 integrin-rich cells in the bulge and the outer cells of ORS were E-cadherin-, β-, and γ-catenin-poor. In the differentiated lanugo hair follicle, bright staining of β1, α2, and α3 integrins was observed in the outer cells of the ORS and was weak in the inner cells of the ORS (Figure 2). α2 subunit is also brightly expressed in the matrix cells of the bulb. β1 and α3 integrins were only weakly expressed in the matrix cells of the bulb. In addition, the matrix cells of the bulb were strongly stained with β- and γ-catenins, although the matrix cells were E-cadherin-poor. Thus, the β1 integrin-rich, E-cadherin-, β-, and γ-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. By the deletion of weak signals using image modification, only the bulge cells and the outer layer cells of the ORS were revealed to exhibit remaining strong signals for β1, α2, and α3 integrins (Figure 3). Image analysis revealed that the bulge region showed weak signals for E-cadherin and β- and γ-catenins. These β1, α2, and α3 integrin-rich cells were E-cadherin-, β-, and γ-catenin-poor. 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 (Figure 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 (Figure 4b). 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 filaments (Figure 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 (Figure 5c,d). Especially, the bulge consists of uniform, small undifferentiated cells. Summarizing the data shown above, possible stem cells, which were β1 integrin-rich, E-cadherin- and β-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 (Figure 6). In this study, unique sequential expression of β1, α2, and α3 integrin subunits, E-cadherin, and β- and γ-catenins were shown at each stage of human hair follicle development. According to the results, strong stainings of β1, α2, and α3 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 α2β1 and α3β1 integrins may be involved in early induction of human hair follicle morphogenesis. Based on the plural evidence that the β1 integrin-rich, E-cadherin-and β- and γ-catenin-poor cells are stem cells of keratinocytes (Molès and Watt, 1997Molès J.P. Watt F.M. The epidermal stem cell compartment: variation in expression levels of E-cadherin and catenins within the basal layer of human epidermis.J Histochem Cytochem. 1997; 45: 867-874Crossref PubMed Scopus (76) Google Scholar), 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 (Figure 6). Our findings 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, 1964Madsen A. Studies on the ''bulge'' (Wulst) in superficial basal cell epithelioma.Arch Dermatol. 1964; 89: 698-708Crossref PubMed Scopus (19) Google Scholar) consisting of undifferentiated cells that are immunolabeled with an antibody to K19 (Lane et al., 1985Lane E.B. Bártek J. Purkis P.E. Leigh I.M. Keratin antigens in differentiating skin.Ann NY Acad Sci. 1985; 455: 241-258Crossref PubMed Scopus (276) Google Scholar;Akiyama et al., 1995Akiyama M. Dale B.A. Sun T.-T. Holbrook K.A. Characterization of hair follicle bulge in human fetal skin; the human fetal bulge is a pool of undifferentiated keratinocytes.J Invest Dermatol. 1995; 105: 844-850Crossref PubMed Scopus (81) Google Scholar), a possible stem cell marker (Lane et al., 1991Lane E.B. Wilson C.A. Hughes B.R. Leigh I.M. Stem cells in hair follicles: cytoskeletal studies.Ann NY Acad Sci. 1991; 642: 197-213Crossref PubMed Scopus (84) Google Scholar;Michel et al., 1996Michel M. Torok N. Godbout M.J. 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. 1996; 109: 1017-1028PubMed Google Scholar). Keratin 15-positive, β1 integrin bright cells were identified in adult human hair follicle bulges (Lyle et al., 1998Lyle S. Christofidou-Solomidou M. Liu Y. Elder D.E. Albelda S. Cotsarelis G. The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells.J Cell Sci. 1998; 111: 3179-3188PubMed Google Scholar). In addition, label-retaining cells were found in the adult human hair follicle bulge (Lyle et al., 1998Lyle S. Christofidou-Solomidou M. Liu Y. Elder D.E. Albelda S. Cotsarelis G. The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells.J Cell Sci. 1998; 111: 3179-3188PubMed Google Scholar). The human fetal bulge cells are also known to strongly express EGF receptor (Akiyama et al., 1996Akiyama M. Smith L.T. Holbrook K.A. Growth factor and growth factor receptor localization in the hair follicle bulge and associated tissue in human fetus.J Invest Dermatol. 1996; 106: 391-396Crossref PubMed Scopus (78) Google Scholar). These observations were confirmed 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 β1 integrin-rich, E-cadherin-, and β- and γ-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 β1 integrin in vivo and in culture is confined to the basal cells in contact with basement membrane (Kaufmann et al., 1989Kaufmann R. Frösch D. Westphal C. Weber L. Klein C.E. Integrin VLA-3: ultrastructural localization at cell-cell contact sites of human cell cultures.J Cell Biol. 1989; 109: 1807-1815Crossref PubMed Scopus (103) Google Scholar;De Luca et al., 1990De Luca M. Tamura R.N. Kajiji S. et al.Polarized integrin mediates human keratinocyte adhesion to basal lamina.Proc Natl Acad Sci USA. 1990; 87: 6888-6892Crossref PubMed Scopus (182) Google Scholar;Hertle et al., 1991Hertle M.D. Adams J.C. Watt F.M. Integrin expression during human epidermal development in vivo and in vitro.Development. 1991; 112: 193-206PubMed Google Scholar;Cerri et al., 1994Cerri A. Favre A. Giunta M. Corte G. Grossi C.E. Berti E. Immunohistochemical localization of a novel β1 integrin in normal and pathologic squamous epithelia.J Invest Dermatol. 1994; 102: 247-252Abstract Full Text PDF PubMed Google Scholar). In adult human anagen hair follicles, a shift in expression of α2β1 and α3β1 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, 1997Commo S. Bernard B.A. The distribution of α2β1 α3β1 and α6β4 integrins identifies distinct subpopulations of basal keratinocytes in the outer root sheath of the human anagen hair follicle.Cell Mol Life Sci. 1997; 53: 466-471Crossref PubMed Scopus (30) Google Scholar). These altered localization patterns may reflect the two different functions of α2β1 and α3β1 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 stainings for β1, α2, and α3 integrin subunits. In addition, the fetal bulge cells exhibited poor expression of E-cadherin and β- and γ-catenins. 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., 1995Carroll J.M. Romero M.R. Watt F.M. Suprabasal integrin expression in the epidermis of transgenic mice results in developmental defects and a phenotype resembling psoriasis.Cell. 1995; 83: 957-968Abstract Full Text PDF PubMed Scopus (277) Google Scholar established β1 integrin transgenic mice using the involucrin promoter that express functional human β1 integrin in the suprabasal epidermal layers. The β1 integrin transgenic mice showed abnormalities of the hairs of the coat and whiskers. The abnormal coat phenotype was apparent as soon as the first coat developed, and there was a progressive normalization through successive hair growth cycles. These findings suggest that regulated β1 integrin expression is essential for the hair follicle development, although the exact mechanisms of functions of β1 integrins for the hair follicle morphogenesis are still unknown. In this context, our findings of ontogenic localization of β1 integrin expression is important to elucidate the roles of β1 integrins in human hair follicle development. In this study, the selected criteria for follicle stem cell stainings, β1 integrins high, E-cadherin-low, β- and γ-catenins-low, were extrapolated from work with epidermal stem cell (Molès and Watt, 1997Molès J.P. Watt F.M. The epidermal stem cell compartment: variation in expression levels of E-cadherin and catenins within the basal layer of human epidermis.J Histochem Cytochem. 1997; 45: 867-874Crossref PubMed Scopus (76) Google Scholar). 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, β- and γ-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., 1990Cotsarelis G. Sun T.-T. Lavker R.M. Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis.Cell. 1990; 61: 1329-1337Abstract Full Text PDF PubMed Scopus (1803) Google Scholar). 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 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 specific 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. 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 fine 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.