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REVIEW ARTICLE A Comprehensive Guide for the Recognition and Classification of Distinct Stages of Hair Follicle Morphogenesis Ralf Paus,¶ Sven Mu¨ller-Ro¨ver,* Carina van der Veen, Marcus Maurer,§ Stefan Eichmu¨ller,† Gao Ling,‡ Udo Hofmann,† Kerstin Foitzik,¶ Lars Mecklenburg,¶** and Bori Handjiski Department of Dermatology, Charite´, Humboldt University, Berlin, and ¶University Hospital Eppendorf, University of Hamburg, Hamburg, Germany; *Center for Cutaneous Research, Queen Mary and Westfield College, London, U.K.; †Clinical Cooperation Unit for Dermato-Oncology (DKFZ), Department of Dermatology, Klinikum Mannheim, University of Heidelberg, Mannheim, Germany; ‡Department of Pathology, Uppsala University Hospital, Uppsala, Sweden; §Department of Pathology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, U.S.A.; **Department of Pathology, School of Veterinary Medicine, Hannover, Germany Numerous spontaneous and experimentally induced mouse mutations develop a hair phenotype, which is often associated with more or less discrete abnorm- alities in hair follicle development. In order to recog- nize these, it is critically important to be able to determine and to classify accurately the major stages of normal murine hair follicle morphogenesis. As an aid, we propose a pragmatic and comprehensive guide, modified after previous suggestions by Hardy, and provide a list of easily recognizable classification cri- teria, illustrated by representative micrographs. Basic and more advanced criteria are distinguished, the former being applicable to all mouse strains and requiring only simple histologic stains (hematoxylin and eosin, Giemsa, periodic acid Schiff, alkaline phos- phatase activity), the latter serving as auxiliary criteria, which require a pigmented mouse strain (like C57BL/ After decades of relative quiescence, basic and appliedhair research have witnessed an impressive renaissanceover the past 10 y. This has resulted to a major extentfrom the application of modern tools of molecularbiology, and from the use of murine hair research models to the ancient challenge of defining the elusive mechanisms of hair growth control (Stenn et al, 1991; Hardy, 1992; Paus, 1996; Stenn et al, 1996, 1998; Philpott and Paus, 1998; Paus and Cotsarchis, 1999). In particular, an ever-increasing number of spontaneous or experimentally generated mouse mutations has provided invaluable insights into the functional significance of selected gene products in Manuscript received January 5, 1999; revised June 30, 1999; accepted for publication July 5, 1999. Reprint requests to: Department of Dermatology, University Hospital Eppendorf, University of Hamburg, Martinistr. 52, D-20246 Hamburg, Germany. Email: paus@uke.uni-hamburg.de Abbreviations: AP, alkaline phosphatase; DP, dermal papilla; HF, hair follicle; IL-1-RI, interleukin 1 receptor type 1; IR, immunoreactivity; IRS, inner root sheath; NCAM, neural cell-adhesion molecule; ORS outer root sheath; PAS, periodic acid Schiff reaction; p.p. post partum (5 days of postnatal life); SG, sebaceous gland; TGF-β-RII, transforming growth factor-β receptor type II. 0022-202X/99/$14.00 · Copyright © 1999 by The Society for Investigative Dermatology, Inc. 523 6J) or immunohistochemistry (interleukin-1 receptor type I, transforming growth factor-b receptor type II). In addition, we present simplified, computer- generated schematic drawings for the standardized recording and reporting of gene and antigen expression patterns during hair follicle development. This classi- fication aid serves as a basic introduction into the field of hair follicle morphogenesis, aims at standardizing the presentation of related hair research data, and should become a useful tool when screening new mouse mutants for discrete abnormalities of hair foll- icle morphogenesis (compared with the respective wild type) in a highly reproducible, easily applicable, and quantifiable manner. Key words: alkaline phosphatase/ C57BL/6J mouse/dermal papilla/interleukin-1 receptor/ transforming growth factor-b receptor. J Invest Dermatol 113:523–532, 1999 the control of hair follicle (HF) morphogenesis and cycling (Sund- berg, 1994; Stenn et al, 1996; Paus and Cotsarchis, 1999; Philpott and Paus, 1998). Often enough, however, the published analyses of the hair phenotype of a new mouse mutation, from a hair researcher’s point of view, are highly unsatisfactory because they tend to be very superficial, inaccurate, and/or incomplete. One basic, but essential requirement for any researcher with an interest in HF biology, is to acquire the ability to recognize and classify accurately the various stages of murine HF development (morphogenesis) (Hardy, 1992; Philpott and Paus, 1998). Unfortun- ately, despite a large body of literature on selected aspects of murine HF development, dating back almost a century (Oyama, 1904; Dry, 1926; Gibbs, 1941; Hardy, 1949; Butcher, 1951; Chase, 1951; Davidson and Hardy, 1952), there is not a single pragmatic guide that comprehensively andquickly introducesnewcomers to the intricacies of HF classification without the need to first digest dozens of studies, each of which covers only selected aspects of HF morphogenesis. This study strives to provide such a guide, which does not require any prior knowledge of HF biology (useful introductions into the latter may be found, for example, in Hardy, 1992; Paus, 1996; Stenn et al, 1996, 1998; Paus and Cotsarchis, 1999). This comprehensive guide to HF morphogenesis aims at standardizing the presentation of hair research data, and should become a useful tool when screening 524 PAUS ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Table I. Glossary of anatomical terms frequently used in hair researcha Term Definition Bulb Prominent, onion-shaped thickening on the proximal end of the HF, consisting of relatively undifferentiated matrix cells, HF melanocytes, as well as of proximal ORS cells Bulbous peg (syn.: Bulbuszapfen) Elongated and more differentiated column of epithelial cells with massive bulb-like aggregation of matrix keratinocytes on the proximal end, recognizable parts of IRS, egg-shaped DP and two solid swellings on the posterior side as a hair bulge and an early sebaceous gland Bulge (syn.: Wulst) Convex extension on the distal part of the ORS, near the epidermis, seat of epithelial follicle stem cells and point of insertion of the muscle arrector pili Dermal papilla (DP) A mesodermal part of the HF, which consists of closely packed mesenchymal cells, framed by the enlarged bulb matrix on the distal end Hair canal Tube-like connection between epidermal surface and most distal part of the IRS, demarcated by surrounding ORS. Hair cone First recognizable part of IRS (pale epithelial layer or Henle’s layer) which starts to develop as a cone-shaped structure above the DP Hair germ (syn.: placode, Haarkeim) Bud-like thickening of the pre-germ plaque in the epidermis consisting of elongated keratinocytes whose proximal end is capped by numerous aggregated mesenchymal cells in the dermis Hair peg (syn.: Haarzapfen) Solid column of epithelial keratinocytes growing into the dermis with a concave proximal end which surrounds partially a compact ball of mesenchymal cells of the future DP Hair shaft The hair per se, composed of trichocytes (5 terminally differentiated HF keratinocytes), organized in the form of hair cuticle, cortex and medulla, and surrounded by the IRS (or ORS at the level of the hair canal) Infundibulum Most distal part of the HF including hair canal and distal ORS, limited proximally by the duct of SG and laterally by cells of the ORS Inner root sheath (IRS) Multilayered structure composed of terminally differentiated HF keratinocytes surrounded by the ORS, consisting of the pale epithelial layer or Henle’s layer as well as Huxley’s layers and cuticle, which covers the hair shaft up to the hair canal. Isthmus Small part of the HF between insertion of the sebaceous gland and the bulge Outer root sheath (ORS) Outermost sheath of HF keratinocytes, which merges distally into the basal layer of epidermis and proximally into the hair bulb; its distal part is often subdivided into a supra- and infrainfundibular portion (see: infundibulum) Pre-germ (syn.: primitive hair germ) Plaque of epidermal cells, recognizable as a ‘‘crowding of nuclei’’ in the basal layer of the epidermis aFor introductory references, see: Sto¨hr, 1903; Oyama, 1904; Pinkus, 1910; Dry, 1926; Hentschel, 1930; Hardy, 1949, 1951; Fleischhauer, 1953; Montagna and van Scott, 1958; Pinkus, 1958; Straile et al, 1961; Parakkal, 1969a; Cotsarelis et al, 1990; Holbrook and Minami, 1991; Abell, 1994; Paus et al, 1994b; Paus and Cotsarchis, 1999 new mouse mutants for discrete abnormalities of HF morphogenesis (compared with the respective wild type) in a highly reproducible, easily applicable, and quantifiable manner. It uses only simple histo- chemical and widely available immunohistologic staining techniques [hematoxylin–eosin, periodic acid Schiff (PAS) reaction, Oil Red O, alkaline phosphatase activity (AP); interleukin-1 receptor type 1 (IL-1-RI), TGF-β receptor type II (TGF-β-RII)]. In addition, we suggest some slightmodificationsofpreviouslyproposedclassification schemes (e.g., Sto¨hr, 1903; Hardy, 1949, 1951; Pinkus, 1958; Hardy, 1992). Among the many different HF subtypes (e.g., vibrissae, tylotrich, and non-tylotrich pelage HF) (Sundberg, 1994), this guide is focused exclusively on murine non-tylotrich pelage follicles, which comprise the vast majority of the truncal fur coat of mice, and most of which develop in the perinatal period (Vielkind et al, 1995; Vielkind and Hardy, 1996; Paus et al, 1997). Nevertheless, the current guide is also broadly applicable to all other HF types seen in any of the haired mammalian species, as they all exhibit the same basic morphologic principles of development. Rather than dealing with fetal HF morphogenesis, this guide depicts neonatal HF development in pigmented C57BL/6J mice, because most researchers will find it easiest to study neonatal rather than fetal HF morphogenesis. Furthermore, we explain how the delicate task of obtaining morphologically satisfactory longitudinal cryosections through the HF can best be mastered, which is essential for a wide range of immunohistochemical studies on murine HF (Fig 1). Finally, a glossary of anatomical terms frequently used in hair research is also provided so as to avoid terminologic confusion (Table I). HOW TO USE THE GUIDE Here, we provide a pragmatic method for obtaining morphologically satisfactory longitudinal cryosections through the HF by using the harvesting and embedding technique developed by U. Hofmann (Paus et al, 1994c) (Fig 1 and legend for details). Furthermore, we provide a comprehensive guide (Fig 2) that is structured as follows: the left-hand column shows a standardized, computer-generated schematic drawing of nine distinct stages of HF morphogenesis (stages 0–8), modified after previous suggestions by Hardy et al (Hardy, 1949, 1951, 1992) (for greater simplicity, we have omitted the subdivision of developmental stage 3 into three separate substages suggested by Hardy). Major anatomical regions of the developing HF are outlined and the corresponding terms are explained in a glossary (Table I). The central column provides a list of easily recognizable classifica- tion criteria, which are then illustrated by three representative micro- graphs of non-tylotrich pelage HF from C57BL/6J mouse neonatal dorsal skin, depicted on the right-hand side of Fig 2. Basic and auxiliary criteria are distinguished in the central column (separated from each other by a dotted line; top: basic criteria, bottom: auxiliary criteria). The basic criteria listed are applicable to all mouse strains, as they require only simple histologic stains (Giemsa, PAS, hematoxy- lin–eosin) and are not dependent on the presence of pigment granules (melanin) in the HF. The auxiliary criteria shown here, which further aid in classification, require a pigmented mouse strain (such as C57BL/6J), histochemistry (AP technique) or immunohistochem- istry (IL-1-RI, TGF-β-receptor type II). The figure legend of Fig 2 explains the corresponding (immuno-) histochemical stains, and the day after birth in neonatal C57BL/6J mice when the depicted dorsal skin samples were harvested. BASIC AND AUXILIARY CRITERIA FOR THE RECOGNITION AND CLASSIFICATION OF DISTINCT STAGES OF HF MORPHOGENESIS HF length During the progression of HF development, the length of developing HF is precisely coupled to the stage of VOL. 113, NO. 4 OCTOBER 1999 HAIR FOLLICLE MORPHOGENESIS GUIDE 525 Figure 1. Embedding procedure for obtaining longitudinal cryosections of mouse pelage HF. As developed by U. Hofmann (Paus et al, 1994c). Tissue banks used for this guide were prepared from neonatal dorsal skin of C57BL/6J mice obtained from Charles River (Sulzfeld, Germany) as described (Paus et al, 1997; Botchkarev et al, 1998a). Skin samples (1 3 3 cm) were obtained from a well-defined dorsal skin region precisely located above the spinal cord at the thoracolumbar region with each end at the same distance (µ1.5 cm) from the smallest part of the neck and the insertion of the tail, respectively. Dorsal skin has been dissected at the level of the subcutis just below the panniculus carnosus (5 subcutaneous muscle layer). For routine histology, biopsies from the upper half of the dissected dorsal skin were cut parallel to the spine to obtain longitudinal HF sections (steps 1–3). After step 6 [freezing the tissue into liquid nitrogen (N2)] it is of crucial importance to avoid any thawing of the skin sample by dipping it again in liquid nitrogen after every consecutive step (steps 7, 9, 11). Thawing inevitably leads to freezing–thawing artifacts such as fast protein degradation, antigen masking, and increased background during immunohistochemistry. 526 PAUS ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY HF morphogenesis (Fig 3). Assessing the length of the HF allows one to obtain a rough idea on whether the developing HF is in an early, middle, or late stage of its morphogenesis. An additional parameter is the location of the most proximal part of the HF in the dermis (stages 1–5) or in the subcutis (stages 6–8). VOL. 113, NO. 4 OCTOBER 1999 HAIR FOLLICLE MORPHOGENESIS GUIDE 527 Time-scale of HF development in C57BL/6J mice Approxim- ately 10% of murine pelage HF have entered into stage 8 at day 3, about 35% at day 5, and about 100% at day 9 (Paus et al, 1998). The first morphologic signs of synchronized catagen development can be detected about days 17–18 after birth (Gibbs, 1941; Hardy, 1949; Botchkarev et al, 1998a, b; Mu¨ller-Ro¨ver et al, 1998; Paus et al, 1998). 528 PAUS ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Stage-specific criteria for each stage of HF development Stage 0 The first stage of HF development (Fig 2, stage 0), also described as pregerm stage (Pinkus, 1958), is characterized as ‘‘an accumulation of nuclei’’ (Pinkus, 1910, 1958; Fleischhauer, 1953), which is virtually impossible to recognize with routine histologic techniques (Fig 2A). We have previously identified TGF-β-RII immunoreactivity, however, as a useful marker for the pregerm stage of HF development in an otherwise morphologically homogeneous epidermis (Fig 2A): the pregerm stage of HF development can easily be detected as sharply demarcated, strongly TGF-β-RII1 plaques of epidermal keratinocytes in the basal and suprabasal cell layers of the epidermis (Fig 2B) (Paus et al, 1997). Stage 1 Confusingly, stage 1 of HF morphogenesis has been described under various names, such as hair germ (Oyama, 1904; Dry, 1926; Chase, 1951; Holbrook and Minami, 1991), germ plate (Chase, 1951), early hair germ (Pinkus, 1958), follicle plug (Hardy, 1949; Hardy and Lyne, 1956), or placode (Hardy and Vielkind, 1996; Vielkind and Hardy, 1996) (Table I). During stage 1 of Figure 3. Schematic representation of the increasing length of the developing HF and the localization of the most proximal part of the HF in the dermis (stages 1–5) or in the subcutis (stages 6–8) during the progression of HF morphogenesis. Note the developmentally controlled changes in skin thickness and HF angle. Figure 2. A comprehensive guide for the recognition and classification of distinct stages of murine HF morphogenesis. The left-hand column shows a computer-generated schematic drawing of nine distinct stages of HF morphogenesis, modified after Hardy (Hardy, 1949; Hardy, 1951; Hardy, 1992) and Paus et al (1997). The second column summarizes simple basic parameters for HF staging (above dotted line), and auxiliary criteria for more precise staging (below dotted line). Depicted is the development of pigmented non-tylotrich pelage HF in the dorsal skin of neonatal C57BL/6J mice (days 1–8 p.p.; note: the day p.p. listed indicates, when during postnatal skin and HF development the corresponding dorsal skin sample was harvested), which is largely representative of the key developmental steps in the morphogenesis of all HF of any mammalian species. Note: for graphical reasons the angles of the HF to the epidermis have been increased. The following staining techniques were employed: A, C, F, I, L, O, R, U, X: Giemsa staining technique (Romeis, 1991). D, G, J, M, P, S, V, Y: AP technique (Handjiski et al, 1994) as marker of the configuration and localization of the DP fibroblasts. B, E, H, K, Q, Z: TGF-β RII immunoreactivity (Paus et al, 1997). N: IL-1-RI immunoreactivity (Eichmu¨ller et al, 1998). Both markers are used to differentiate the length of the developing TGF-β RII-negative and IL-1-RI -negative IRS framed by the TGF-β-RII1 and IL-1-RI1 keratinocytes of the follicular cord and the ORS. T, W: Oil Red O staining (Romeis, 1991) as marker of the developing SG and the sebum-filled hair canal. Stage 0, day 1 p.p. (A) morphologically homogeneous epidermis (a), no visible follicles (note: at this day p.p., one also finds numerous pelage HF in various stages of their morphogenesis that had already developed in the pre- and perinatal period, as HF development does not appear to be synchronized).(B) the hair germ can only be visualized as well-demarcated plaques of TGF-β-RII1 intraepidermal keratinocytes (b) that are morphologically indistinguishable from their TGF-β-RII-negative neighbors (a). Stage 1, day 1 p.p. (C) early hair germ (a) with aggregation of mesenchymal cells below the epidermal thickening (b). (D) only weakly AP1 mesenchymal cells (d) closely attached to the hair germ (epidermal thickening) (a). (E) the hair germ can only be precisely visualized as TGF-β-RII1 keratinocytes (c) in the basal layer of the epidermis. Stage 2, day 2 p.p. (F) enlarged hair germ (a) with a convex proximal end and closely attached mesenchymal cells (b). (G) AP1 dermal fibroblasts (d) below the elongated hair plug (a). (H) TGF-β-RII IR demarcates the convex end of the hair germ (c). Stage 3, days 2–3 p.p. (I) enlarged hair peg (a) with concave proximal end; the central group of keratinocytes (b) displays a columnar arrangement radially to the follicular axis; the dermal fibroblasts form a rounded, more oval-shaped DP (c). (J) AP staining reveals an oval-shaped DP (e) partially embedded in the concave end of the hair plug (a). (K) IL1-RI IR shows the length of the hair peg (d) and indicates its concave end (a). Stage 4, day 3 p.p. (L) developing bulb (a) and cone-shaped formation of the IRS (b). (M) developing bulb (a) enclosing the AP1 DP (e). (N) IL-1R IR of the developing ORS (d) demarcates the IL-1R -negative DP (c) and the developing IL-1R-negative IRS (b). Stage 5, days 3–4 p.p. (O) elongating IRS (a), developing bulge (b), first sebocytes (c), almost completely enclosed DP (d) and the first melanine granules (h). (p) AP IR reveals that the DP (f) is completely enclosed by hair bulb keratin