Plasticity and Cytokinetic Dynamics of the Hair Follicle Mesenchyme: Implications for Hair Growth Control
2003; Elsevier BV; Volume: 120; Issue: 6 Linguagem: Inglês
10.1046/j.1523-1747.2003.12237.x
ISSN1523-1747
AutoresDesmond J. Tobin, Markus Magerl, Andrei Gunin, Bori Handijski, Ralf Paus,
Tópico(s)Cancer and Skin Lesions
ResumoThe continuously remodeled hair follicle is a uniquely exploitable epithelial-mesenchymal interaction system. In contrast to the cyclical fate of the hair follicle epithelium, the dynamics of the supposedly stable hair follicle mesenchyme remains enigmatic. Here we address this issue using the C57BL/6 hair research model.During hair growth, increase in total follicular papilla size was associated with doubling of papilla cell numbers, much of which occurred before intra-follicular papilla cell proliferation, and subsequent to mitosis in the proximal connective tissue sheath. This indicates that some papilla cells originate in, and migrate from, the proliferating pool of connective tissue sheath fibroblasts. Follicular papilla cell number and total papilla size were maximal by anagen VI, but intriguingly, decreased by 25% during this period of sustained hair production. This cell loss, which continued during catagen, was not associated with intra-follicular papilla apoptosis, strongly indicating that fibroblasts migrate out of the late anagen/early catagen papilla and re-enter the proximal connective tissue sheath. Low-level apoptosis occurred only here, along with the "detachment" of cells from the regressing connective tissue sheath.Thus, the hair follicle mesenchyme exhibits significant hair cycle-associated plasticity. Modulation of these cell interchanges is likely to be important during clinically important hair follicle transformations, e.g. vellus-to-terminal and terminal-to-vellus during androgenetic alopecia. The continuously remodeled hair follicle is a uniquely exploitable epithelial-mesenchymal interaction system. In contrast to the cyclical fate of the hair follicle epithelium, the dynamics of the supposedly stable hair follicle mesenchyme remains enigmatic. Here we address this issue using the C57BL/6 hair research model. During hair growth, increase in total follicular papilla size was associated with doubling of papilla cell numbers, much of which occurred before intra-follicular papilla cell proliferation, and subsequent to mitosis in the proximal connective tissue sheath. This indicates that some papilla cells originate in, and migrate from, the proliferating pool of connective tissue sheath fibroblasts. Follicular papilla cell number and total papilla size were maximal by anagen VI, but intriguingly, decreased by 25% during this period of sustained hair production. This cell loss, which continued during catagen, was not associated with intra-follicular papilla apoptosis, strongly indicating that fibroblasts migrate out of the late anagen/early catagen papilla and re-enter the proximal connective tissue sheath. Low-level apoptosis occurred only here, along with the "detachment" of cells from the regressing connective tissue sheath. Thus, the hair follicle mesenchyme exhibits significant hair cycle-associated plasticity. Modulation of these cell interchanges is likely to be important during clinically important hair follicle transformations, e.g. vellus-to-terminal and terminal-to-vellus during androgenetic alopecia. Complex interactions between ectodermal and mesodermal components of the hair follicle result in the elaboration of five or six concentric cylinders of at least 15 distinct interacting cell subpopulations. These together produce a truly exceptional mini-organ (Paus and Cotsarelis, 1999Paus R. Cotsarelis G. The biology of hair follicles.N Engl J Med. 1999; 341: 491-497Crossref PubMed Scopus (883) Google Scholar) that rivals the vertebrate limb bud (Schaller et al., 2001Schaller S.A. Li S. Ngo-Muller V. Han M.J. Omi M. Anderson R. Muneoka K. Cell biology of limb patterning.Int Rev Cytol. 2001; 203: 483-517Crossref PubMed Scopus (14) Google Scholar) as a model for studies of the genetic regulation of morphogenesis (Philpott and Paus, 1998Philpott M.J. Paus R. Principles of hair follicle morphogenesis.in: Chuong C.-M. Molecular Basis of Epithelial Appendage Morphogenesis. R.G. Landes Company, Austin, Texas1998: 75-103Google Scholar; Cotsarelis and Millar, 2001Cotsarelis G. Millar S.E. Towards a molecular understanding of hair loss and its treatment.Trends Mol Med. 2001; 7: 293-301Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). The hair follicle is unique in the adult mammalian body in experiencing multiple and life-long recapitulations of its embryogenesis whenever it enters into its active growth stage (anagen) (Paus et al., 1999Paus R. Müller-Röver S. Botchkarev V.A. Chronobiology of the hair follicle: Hunting the "hair cycle clock".J Invest Dermatol Symp Proc. 1999; 4: 338-345Abstract Full Text PDF PubMed Scopus (72) Google Scholar; Stenn and Paus, 2001Stenn K.S. Paus R. Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1032) Google Scholar). Critical to the control of this cyclical behavior are the follicular papilla (FP) and connective tissue sheath (CTS), which together form the hair follicle's mesenchymal compartments (Jahoda and Reynolds, 1996Jahoda C.A. Reynolds A.J. Dermal–epidermal interactions. Adult follicle-derived cell populations and hair growth.Dermatol Clin. 1996; 4: 573-583Abstract Full Text Full Text PDF Scopus (111) Google Scholar). Previous studies have convincingly demonstrated that FP fibroblasts, and the morphogens they secrete, are critical in hair growth induction (anagen) (Cohen, 1961Cohen J. Transplantation of individual rat and guinea pig whisker papilla.J Embryol Exp Morph. 1961; 9: 117-127PubMed Google Scholar; Oliver, 1967Oliver R.F. The experimental induction of whisker growth in the hooded rat by implantation of dermal papillae.J Embryol Exp Morph. 1967; 18: 43-51PubMed Google Scholar; Jahoda et al., 1984Jahoda C.A. Horne K.A. Oliver R.F. Induction of hair growth by implantation of cultured dermal papilla cells.Nature. 1984; 311: 560-562Crossref PubMed Scopus (466) Google Scholar; Reynolds et al., 1991Reynolds A.J. Oliver R.F. Jahoda C.A. Dermal cell populations show variable competence in epidermal cell support: Stimulatory effects of hair papilla cells.J Cell Sci. 1991; 98: 75-83PubMed Google Scholar; Robinson et al., 2001Robinson M. Reynolds A.J. Gharzi A. Jahoda C.A. In vivo induction of hair growth by dermal cells isolated from hair follicles after extended organ culture.J Invest Dermatol. 2001; 117: 596-604Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Moreover, these components are critical for determining the developmental pathways of the overlying ectodermal cell lineages during hair follicle morphogenesis and cycling (Philpott and Paus, 1998Philpott M.J. Paus R. Principles of hair follicle morphogenesis.in: Chuong C.-M. Molecular Basis of Epithelial Appendage Morphogenesis. R.G. Landes Company, Austin, Texas1998: 75-103Google Scholar; Matsuzaki and Yoshizato, 1998Matsuzaki T. Yoshizato K. Role of hair papilla cells on induction and regeneration processes of hair follicles.Wound Repair Regen. 1998; 6: 524-530Crossref PubMed Scopus (47) Google Scholar; Kishimoto et al., 2000Kishimoto J. Burgeson R.E. Morgan B.A. Wnt signaling maintains the hair-inducing activity of the dermal papilla.Genes Dev. 2000; 14: 1181-1185PubMed Google Scholar; Lindner et al., 2000Lindner G. Menrad A. Gherardi E. et al.Involvement of hepatocyte growth factor/scatter factor and met receptor signaling in hair follicle morphogenesis and cycling.FASEB J. 2000; 14: 319-332Crossref PubMed Scopus (110) Google Scholar; Langbein et al., 2001Langbein L. Rogers M.A. Winter H. Praetzel S. Schweizer J. The catalog of human hair keratins. II. Expression of the six type II members in the hair follicle and the combined catalog of human type I and II keratins.J Biol Chem. 2001; 276: 35123-35132Crossref PubMed Scopus (226) Google Scholar), as long as close FP–epithelial contact is maintained (Link et al., 1990Link R.E. Paus R. Stenn K.S. Kuklinska E. Moellmann G. Epithelial growth by rat vibrissae follicles in vitro requires mesenchymal contact via native ECM.J Invest Dermatol. 1990; 95: 202-207Crossref PubMed Scopus (55) Google Scholar; Jahoda and Reynolds, 1996Jahoda C.A. Reynolds A.J. Dermal–epidermal interactions. Adult follicle-derived cell populations and hair growth.Dermatol Clin. 1996; 4: 573-583Abstract Full Text Full Text PDF Scopus (111) Google Scholar). Where this collapses, e.g., in the absence of a functional hr gene product, hair growth is aborted and the hair follicle degenerates (Panteleyev et al., 1998Panteleyev A.A. Paus R. Ahmad W. Sundberg J.P. Christiano A.M. Molecular and functional aspects of the hairless (hr) gene in laboratory rodents and humans.Exp Dermatol. 1998; 7: 249-267Crossref PubMed Scopus (114) Google Scholar). A positive linear relationship has long been recognized to exist between FP volume and hair caliber (Van Scott and Ekel, 1958Van Scott E.J. Ekel T.M. Geometric relationships between the matrix of the hair bulb and its dermal papilla in normal and alopecic scalp.J Invest Dermatol. 1958; 1: 281-287Abstract Full Text PDF Scopus (24) Google Scholar): the volumetric ratio of FP cells (and their secretory activity) to hair matrix keratinocytes is crucially important for determining the size of the hair shaft produced. Additionally, the caliber of the hair shaft can also vary along its length, i.e., fine distal tip, thick mid region, and narrow proximal "club" end (Hutchinson and Thompson, 1997Hutchinson P.E. Thompson J.R. The cross-sectional size and shape of human terminal scalp hair.Br J Dermatol. 1997; 136: 159-165Crossref PubMed Scopus (32) Google Scholar). Thus, this FP/matrix volumetric ratio is likely to change not only between the main stages of the hair cycle, but also during the substages of anagen, as in wool follicles (Ibrahim and Wright, 1982Ibrahim L. Wright E.A. A quantitative study of hair growth using mouse and rat vibrissal follicles. I. Dermal papilla volume determines hair volume.J Embryol Exp Morph. 1982; 72: 209-222PubMed Google Scholar). Studying changes in FP volume and cell number can be expected to provide crucial information on changes in the secretory activity of the FP that underlie all hair follicle transformations during hair follicle cycling, vellus-to-terminal/terminal-to-vellus hair transformation events and changes in the hair shaft diameter, length, and pigmentation (Jahoda and Reynolds, 1996Jahoda C.A. Reynolds A.J. Dermal–epidermal interactions. Adult follicle-derived cell populations and hair growth.Dermatol Clin. 1996; 4: 573-583Abstract Full Text Full Text PDF Scopus (111) Google Scholar; Jahoda, 1998Jahoda C.A. Cellular and developmental aspects of androgenetic alopecia.Exp Dermatol. 1998; 7: 235-248PubMed Google Scholar; Paus et al., 1999Paus R. Müller-Röver S. Botchkarev V.A. Chronobiology of the hair follicle: Hunting the "hair cycle clock".J Invest Dermatol Symp Proc. 1999; 4: 338-345Abstract Full Text PDF PubMed Scopus (72) Google Scholar; Stenn and Paus, 2001Stenn K.S. Paus R. Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1032) Google Scholar). The prevailing consensus in FP cell biology is that the hair follicle mesenchyme represents a very stable cell population with very little if any proliferative activity (Pierard and de la Brassinne, 1975Pierard G.E. de la Brassinne M. Modulation of dermal cell activity during hair growth in the rat.J Cutan Pathol. 1975: 35-41Crossref Scopus (38) Google Scholar; Jahoda, 1998Jahoda C.A. Cellular and developmental aspects of androgenetic alopecia.Exp Dermatol. 1998; 7: 235-248PubMed Google Scholar). This view is supported more recently by the observation that the FP fibroblasts do not undergo apoptosis during hair follicle regression (catagen) (Weedon and Strutton, 1981Weedon D. Strutton G. Apoptosis as the mechanism of the involution of hair follicles in catagen transformation.Acta Derm Venereol. 1981; 61: 335-339PubMed Google Scholar; Lindner et al., 1997Lindner G. Botchkarev V.A. Botchkareva N.V. Ling G. van der Veen C. Paus R. Analysis of apoptosis during hair follicle regression (catagen).Am J Pathol. 1997; 151: 1601-1617PubMed Google Scholar; Stenn et al., 1994Stenn K.S. Lawrence L. Veis D. Korsmeyer S. Seiberg M. Expression of the bcl-2 protooncogene in the cycling adult mouse hair follicle.J Invest Dermatol. 1994; 103: 107-111Crossref PubMed Scopus (90) Google Scholar) as they may be protected from apoptosis via permanent expression of high levels of the anti-apoptotic protein Bcl-2 (Stenn et al., 1994Stenn K.S. Lawrence L. Veis D. Korsmeyer S. Seiberg M. Expression of the bcl-2 protooncogene in the cycling adult mouse hair follicle.J Invest Dermatol. 1994; 103: 107-111Crossref PubMed Scopus (90) Google Scholar; Lindner et al., 1997Lindner G. Botchkarev V.A. Botchkareva N.V. Ling G. van der Veen C. Paus R. Analysis of apoptosis during hair follicle regression (catagen).Am J Pathol. 1997; 151: 1601-1617PubMed Google Scholar). There is, however, substantial clinical evidence that the FP is not static throughout life. This can be easily appreciated from the dramatic increases in FP size and cell number during puberty-associated vellus-to-terminal hair transformation (Barth, 1987Barth J.H. Normal hair growth in children.Pediatr Dermatol. 1987; 4: 173-184Crossref PubMed Scopus (36) Google Scholar), hirsutism, and hypertrichosis, and vice versa during androgenetic alopecia development (Ishino et al., 1997Ishino A. Uzuka M. Tsuji Y. Nakanishi J. Hanzawa N. Imamura S. Progressive decrease in hair diameter in Japanese with male pattern baldness.J Dermatol. 1997; 24: 758-764Crossref PubMed Scopus (23) Google Scholar). Although androgens appear to play a major part, the underlying mechanisms involved in these hair follicle transformations are unclear. In particular, convincing explanations for an increase in FP cell number during the growth phase of the hair cycle have, for the most part, not been found in murine proliferation studies (Wessells and Roessner, 1965Wessells N.K. Roessner K.D. Nonproliferation in dermal condensations of mouse vibrissae and pelage hairs.Dev Biol. 1965; 12: 419-433Crossref PubMed Scopus (62) Google Scholar; Ibrahim and Wright, 1982Ibrahim L. Wright E.A. A quantitative study of hair growth using mouse and rat vibrissal follicles. I. Dermal papilla volume determines hair volume.J Embryol Exp Morph. 1982; 72: 209-222PubMed Google Scholar; Tezuka et al., 1991Tezuka M. Ito M. Ito K. Tazawa T. Sato Y. Investigation of germinative cells in generating and renewed anagen hair apparatus in mice using anti-bromodeoxyuridine monoclonal antibody.J Dermatol Sci. 1991; 2: 434-443Abstract Full Text PDF PubMed Scopus (9) Google Scholar). By contrast, FP cell proliferation has been reported in the primary sheep wool hair follicle (Adelson and Kelley, 1992Adelson D.L. Kelley B.A. Increase in dermal papilla cells by proliferation during development of the primary wool follicle.Aust J Agric Res. 1992; 43: 843-856Crossref Scopus (15) Google Scholar) and in rat pelage follicles (Pierard and de la Brassinne, 1975Pierard G.E. de la Brassinne M. Modulation of dermal cell activity during hair growth in the rat.J Cutan Pathol. 1975: 35-41Crossref Scopus (38) Google Scholar). It remained unclear, however, whether this intrapapillary cell proliferation indeed reflected proliferating fibroblasts, as other potential sources of cell division include the endothelium of intrapapillary capillaries (Pierard and de la Brassinne, 1975Pierard G.E. de la Brassinne M. Modulation of dermal cell activity during hair growth in the rat.J Cutan Pathol. 1975: 35-41Crossref Scopus (38) Google Scholar). In contrast to the FP, the relative contribution of the follicular CTS to hair growth control has only rather recently become systematically investigated (Reynolds et al., 1991Reynolds A.J. Oliver R.F. Jahoda C.A. Dermal cell populations show variable competence in epidermal cell support: Stimulatory effects of hair papilla cells.J Cell Sci. 1991; 98: 75-83PubMed Google Scholar; Jahoda et al., 1991Jahoda C.A. Reynolds A.J. Chaponnier C. Forester J.C. Gabbiani G. Smooth muscle alpha-actin is a marker for hair follicle dermis in vivo and in vitro.J Cell Sci. 1991; 99: 627-636PubMed Google Scholar; Horne and Jahoda, 1992Horne K.A. Jahoda C.A. Restoration of hair growth by surgical implantation of follicular dermal sheath.Development. 1992; 116: 563-571PubMed Google Scholar; Matsuzaki et al., 1996Matsuzaki T. Inamatsu M. Yoshizato K. The upper dermal sheath has a potential to regenerate the hair in the rat follicular epidermis.Differentiation. 1996; 60: 287-297Crossref PubMed Google Scholar; Jahoda, 1998Jahoda C.A. Cellular and developmental aspects of androgenetic alopecia.Exp Dermatol. 1998; 7: 235-248PubMed Google Scholar). Early indication of a fundamental interaction between these two mesenchymal components of the hair follicle was convincingly demonstrated by the reformation of a FP from the lower CTS (Oliver, 1967Oliver R.F. The experimental induction of whisker growth in the hooded rat by implantation of dermal papillae.J Embryol Exp Morph. 1967; 18: 43-51PubMed Google Scholar), indicating that significant plasticity exists within these two hair follicle fibroblast subpopulations, at least under conditions of experimental manipulation/trauma. Cell renewal in the CTS is also thought to be a very rare event. Indeed, the number of resident fibroblasts has been claimed to remain constant during the entire hair growth cycle (Pierard and de la Brassinne, 1975Pierard G.E. de la Brassinne M. Modulation of dermal cell activity during hair growth in the rat.J Cutan Pathol. 1975: 35-41Crossref Scopus (38) Google Scholar). Despite the marked hair growth cycle-dependent changes in hair follicle epithelial and mesenchymal cellularity (Parakkal, 1990Parakkal P.F. Catagen and telogen phases of the growth cycle.in: Orfanos C.E. Happle R. Hair and Hair Diseases. Springer-Verlag, Berlin1990: 99-116Crossref Google Scholar; Jahoda et al., 1992Jahoda C.A. Mauger A. Bard S. Sengel P. Changes in fibronectin, laminin and type IV collagen distribution relate to basement membrane restructuring during the rat vibrissa follicle hair growth cycle.J Anat. 1992; 181: 47-60PubMed Google Scholar) there is no consensus on the actual mechanism of CTS regression, including the fate of its cells and basal lamina/glassy membrane (Montagna and Parakkal, 1974Montagna W. Parakkal P.F. The Structure and Function of Skin. Academic Press, New York1974Google Scholar). On this background, we have employed the most comprehensively studied animal model in hair research, the C57BL/6 mouse (Paus et al., 1990Paus R. Stenn K.S. Link R.E. Telogen skin contains an inhibitor of hair growth.Br J Dermatol. 1990; 122: 777-784Crossref PubMed Scopus (208) Google Scholar), and have used both the depilation-induced and the spontaneous murine hair cycle to analyze systematically the plasticity and dynamics of the FP and CTS during hair follicle cycling. Another important advantage of using this model is that the absence of capillaries in their pelage FP (Durward and Rudall, 1958Durward A. Rudall K.M. The vascularity and patterns of growth of hair follicles.in: Montagna W. Ellis R.A. The Biology of Hair Growth. Academic Press, New York1958: 469-485Crossref Google Scholar) avoids the ambiguity affecting the interpretation of proliferating cells in the vascularized human hair follicle. The following specific questions were addressed: (i) What drives the reconstruction of the hair follicle mesenchyme during early anagen? (ii) Is the anagen-associated increase in FP size due to cell proliferation, cell migration, cell growth, and/or increases in production of extracellular matrix (ECM)? (iii) How does the reconstructing hair follicle epithelium influence the organization of the hair follicle mesenchyme during anagen development? (iv) Does regression of the hair follicle mesenchyme during catagen involve FP cell emigration to the CTS, loss of FP cell synthetic activity, and/or mesenchymal cell death? The questions were addressed by histomorphometric analyses of a range of proliferation markers, and planimetric analysis of changes in FP size, cell volume, and cell number. Cellular activity was examined using high-resolution light microscopy and transmission electron microscopy (TEM). Female syngeneic C57BL/6 mice (Charles River, Sulzfeld, Germany) were used that had all back skin hair follicles in either the resting phase of the hair cycle (telogen; 6–9 wk of age) or had hair follicles that had just entered the first true cycle (anagen; postpartum day 28) after having completed hair follicle morphogenesis (Paus et al., 1999Paus R. Müller-Röver S. Botchkarev V.A. Chronobiology of the hair follicle: Hunting the "hair cycle clock".J Invest Dermatol Symp Proc. 1999; 4: 338-345Abstract Full Text PDF PubMed Scopus (72) Google Scholar; Müller-Röver et al., 2001Müller-Röver S. Handjiski B. van der Veen C. Eichmuller S. Foitzik K. McKay I.A. Stenn K.S. Paus R. A comprehensive guide for the accurate classification of murine hair follicles in district hair cycle stages.J Invest Dermatol. 2001; 117: 3-15Abstract Full Text Full Text PDF PubMed Google Scholar). The animals were housed in community cages with 12 h light periods, and were fed water and mouse chow ad libitum. Hair follicle cycling was synchronized by wax/rosin depilation to obtain large numbers of hair follicles in the same hair cycle stage as previously described (Paus et al., 1990Paus R. Stenn K.S. Link R.E. Telogen skin contains an inhibitor of hair growth.Br J Dermatol. 1990; 122: 777-784Crossref PubMed Scopus (208) Google Scholar). Back skin was harvested on days 0, 1, 3, 5, 8, 12, 14, 15, 16, 17, 18, 19, 20, 25, and 34 postdepilation. In this way tissue was collected that contained hair follicles as they passed through one hair cycle from telogen (resting phase) to the start of hair regrowth (anagen I), via active hair shaft production (anagen IV–VI), through apoptosis-driven hair follicle regression (catagen), back to telogen (Müller-Röver et al., 2001Müller-Röver S. Handjiski B. van der Veen C. Eichmuller S. Foitzik K. McKay I.A. Stenn K.S. Paus R. A comprehensive guide for the accurate classification of murine hair follicles in district hair cycle stages.J Invest Dermatol. 2001; 117: 3-15Abstract Full Text Full Text PDF PubMed Google Scholar). Immediately upon removal, the tissue was divided; part snap frozen and acetone fixed for Ki67 and proliferating cell nuclear antigen (PCNA) immunohistochemistry (Magerl et al., 2001Magerl M. Tobin D.J. Müller-Röver S. Hagen E. Lindner G. McKay I. Paus R. Patterns of proliferation and apoptosis during murine hair follicle morphogenesis.J Invest Dermatol. 2001; 116: 947-955Abstract Full Text Full Text PDF PubMed Google Scholar) and part fixed in Karnovsky's fixative for high-resolution light microscopy and electron microscopy (Tobin et al., 1998Tobin D.J. Hagen E. Botchkarev V.A. Paus R. Do hair bulb melanocytes undergo apoptosis during hair follicle regression (catagen)?.J Invest Dermatol. 1998; 111: 941-947Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Skin treated with bromodeoxyuridine (BrdU) and colchicine (see below) for the assessment of mitotic indices was prepared as for Ki67 and PCNA. In addition, back skin of 28 d old neonatal mice was harvested tissue and was assessed by high-resolution light microscopy and TEM as described bellow. This tissue contains hair follicles passing from the end of morphogenesis to the start of hair regrowth (anagen I), i.e., entering the first true anagen phase of the first cycle, and so facilitated a direct comparison between depilation-induced and spontaneous anagen. All animal studies were performed as approved by the responsible government institution, Berlin. Cell proliferation was assessed via the immunohistochemical detection of two proliferation-associated proteins. Seven micrometer paraffin-embedded sections were incubated with the proliferation marker antibodies. The monoclonal antibody Ki67 (Dianova, GmbH, Hamburg, Germany) was employed to detect a 345–395 kDa protein complex expressed primarily during the S, G2, and M phases of the cell cycle. Additionally, a monoclonal antibody to PCNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to detect an auxiliary protein to DNA polymerase (present in cycling cells and most abundant during S phase. Following primary antibody incubations, sections were treated with peroxidase-labeled secondary antibody (Jackson Immuno-Research Labs, Inc., West Grove, PA, USA) and developed using 3,3′-diaminobenzidine-tetrachloride chromogen as previously described (Magerl et al., 2001Magerl M. Tobin D.J. Müller-Röver S. Hagen E. Lindner G. McKay I. Paus R. Patterns of proliferation and apoptosis during murine hair follicle morphogenesis.J Invest Dermatol. 2001; 116: 947-955Abstract Full Text Full Text PDF PubMed Google Scholar). Cell proliferation was further assessed by BrdU incorporation into cycling hair follicle cells to identify adequately cells in the S phase of the cell cycle, as previously described (Tezuka et al., 1991Tezuka M. Ito M. Ito K. Tazawa T. Sato Y. Investigation of germinative cells in generating and renewed anagen hair apparatus in mice using anti-bromodeoxyuridine monoclonal antibody.J Dermatol Sci. 1991; 2: 434-443Abstract Full Text PDF PubMed Scopus (9) Google Scholar). Briefly, mice were injected intraperitoneally with BrdU (Sigma-Aldrich Chemie GmbH, Munich, Germany) dissolved in saline (20 microgram/g BW) 3 h before being killed. Harvested tissue was fixed in 4% formaldehyde and paraffin embedded. BrdU incorporation during S phase was examined immunohistochemically using anti-BrdU antibody (Santa Cruz), and alkaline phosphatase secondary antibody (Jackson Immuno-Research Labs). Color was developed with naphthol AS-BI phosphate/new fuchsin and hematoxylin as counterstain. In order to identify and quantify cells in the M phase of the cell cycle, selected mice were injected with colchicine (0.1 mg in 250 μl saline) 3 h before euthanasia. Thereafter, skin specimens were fixed and processed as above, counter-stained with Weigert's iron hematoxylin (for highlighting mitotic figures), and the number of cells with visible, colchicine-arrested, mitotic spindles counted in the hair follicle mesenchyme. Incidence of apoptosis was assessed using classical morphologic criteria by both high-resolution light microscopy and TEM (Magerl et al., 2001Magerl M. Tobin D.J. Müller-Röver S. Hagen E. Lindner G. McKay I. Paus R. Patterns of proliferation and apoptosis during murine hair follicle morphogenesis.J Invest Dermatol. 2001; 116: 947-955Abstract Full Text Full Text PDF PubMed Google Scholar). This was systematically compared with previously published results on mesenchymal cell apoptosis using the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling) technique (Lindner et al., 1997Lindner G. Botchkarev V.A. Botchkareva N.V. Ling G. van der Veen C. Paus R. Analysis of apoptosis during hair follicle regression (catagen).Am J Pathol. 1997; 151: 1601-1617PubMed Google Scholar; Magerl et al., 2001Magerl M. Tobin D.J. Müller-Röver S. Hagen E. Lindner G. McKay I. Paus R. Patterns of proliferation and apoptosis during murine hair follicle morphogenesis.J Invest Dermatol. 2001; 116: 947-955Abstract Full Text Full Text PDF PubMed Google Scholar). Representative tissue samples of skin from three mice at each of the test days above and from three p28 (postpartum day 28) mice were fixed in Karnovsky's fixative, post-fixed in 2% osmium tetroxide and uranyl acetate, and embedded in resin as previously described (Tobin et al., 1998Tobin D.J. Hagen E. Botchkarev V.A. Paus R. Do hair bulb melanocytes undergo apoptosis during hair follicle regression (catagen)?.J Invest Dermatol. 1998; 111: 941-947Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Semithin and ultrathin sections were cut with a Reichart-Jung microtome (Leica GmbH, Wetzlar, Germany); the former were stained with the metachromatic stain, toluidine blue/borax, examined by light microscopy and photographed (Leitz, Germany). Ultrathin sections were stained with uranyl acetate and lead citrate and were examined and photographed using a JEM-1200EX (Jeol, Tokyo, Japan). electron microscope. For quantitative histomorphometry, the hair follicle mesenchyme was divided into two defined subcompartments: the entire FP, and the proximal CTS. PCNA, Ki67, and BrdU proliferation indices were calculated as the number of labeled cells per 100 cells. The mitotic index was calculated in colchicine-treated specimens as a percentage of mitotic cells (anaphase to telophase) per total cells in the hair follicle compartment under view. In all cases, no less than 1000 cells in CTS were viewed per mouse. Twenty hair follicles in each of three blocks from three mice and at all time points were examined by light microscopy (i.e., total 2880 hair follicles). Five hair follicles from each block from each of three mice were examined by TEM at each test day (i.e., total 720 hair follicles). Arithmetic means and standard error of the mean were calculated. Statistical comparisons were performed with one-way analysis of variance and post-hoc Tukey's test (Statistica 5.0, StatSoft Inc.). Cells located in the proximal CTS of telogen hair follicle undergo an abrupt and dramatic re-entry into the cell cycle approximately 1 d after anagen induction by depilation (Figure 1a). As early as anagen I/II, up to 12% of cells in this component of the hair follicle mesenchyme are proliferating. By contrast, no proliferating cells were detected within the FP at this time. As anagen development progressed, the mitotic index in the proximal CTS peaked at 30%, whereas the mitotic index within the FP was never above 5% (Figure 1b)
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