A Comprehensive Guide for the Recognition and Classification of Distinct Stages of Hair Follicle Morphogenesis

Numerous spontaneous and experimentally induced mouse mutations develop a hair phenotype, which is often associated with more or less discrete abnormalities in hair follicle development. In order to recognize 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 criteria, 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 phosphatase activity), the latter serving as auxiliary criteria, which require a pigmented mouse strain (like C57BL/6J) or immunohistochemistry (interleukin-1 receptor type I, transforming growth factor-β 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 classification 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 follicle morphogenesis (compared with the respective wild type) in a highly reproducible, easily applicable, and quantifiable manner. Numerous spontaneous and experimentally induced mouse mutations develop a hair phenotype, which is often associated with more or less discrete abnormalities in hair follicle development. In order to recognize 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 criteria, 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 phosphatase activity), the latter serving as auxiliary criteria, which require a pigmented mouse strain (like C57BL/6J) or immunohistochemistry (interleukin-1 receptor type I, transforming growth factor-β 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 classification 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 follicle morphogenesis (compared with the respective wild type) in a highly reproducible, easily applicable, and quantifiable manner. alkaline phosphatase dermal papilla hair follicle interleukin 1 receptor type 1 immunoreactivity inner root sheath neural cell-adhesion molecule outer root sheath periodic acid Schiff reaction post partum (= days of postnatal life) sebaceous gland transforming growth factor-β receptor type II After decades of relative quiescence, basic and applied hair research have witnessed an impressive renaissance over the past 10 y. This has resulted to a major extent from the application of modern tools of molecular biology, 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., 1991Stenn K.S. Messenger A.G. Baden H.P. The molecular and structural biology of hair.Ann N Y Acad Sci. 1991; 642: 1-519Google Scholar;Hardy, 1992Hardy M.H. The secret life of the hair follicle.Trends Genet. 1992; 8: 55-61Abstract Full Text PDF PubMed Scopus (762) Google Scholar;Paus, 1996Paus R. Control of the hair cycle and hair diseases as cycling disorders.Curr Opin Dermatol. 1996; 3: 248-258Google Scholar;Stenn et al., 1996Stenn K.S. Combates N.J. Eilertsen K.J. Gordon J.S. Pardinas J.R. Parimoo S. Prouty S.M. Hair follicle growth controls.Dermatol Clinics. 1996; 14: 543-558Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar,Stenn et al., 1998Stenn K. Parimoo S. Prouty S. Growth of the hair follicle: A cycling and regenerating biological system.in: Chuong C.M. Molecular Basis of Epithelial Appendage Morphogenesis. Landes Bioscience Publishers, Austin, TX1998: 111-124Google Scholar;Philpott and Paus, 1998Philpott M.P. Paus R. Principles of hair follicle morphogenesis.in: Chuong C.M. Molecular Basis of Epithelial Appendage Morphogenesis. Landes Bioscience Publishers, Austin, TX1998: 75-103Google Scholar;Paus & Cotsarchis, 1999Paus R. Cotsarchis G. The biology of hair follicles.N Engl J Med. 1999; 341: 491-497Crossref PubMed Scopus (883) Google Scholar). 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 the control of hair follicle (HF) morphogenesis and cycling (Sundberg, 1994Sundberg J.P. Handbook of Mouse Mutations with Skin and Hair Abnormalities. CRC Press, Boca Raton1994Google Scholar;Stenn et al., 1996Stenn K.S. Combates N.J. Eilertsen K.J. Gordon J.S. Pardinas J.R. Parimoo S. Prouty S.M. Hair follicle growth controls.Dermatol Clinics. 1996; 14: 543-558Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar;Paus & Cotsarchis, 1999Paus R. Cotsarchis G. The biology of hair follicles.N Engl J Med. 1999; 341: 491-497Crossref PubMed Scopus (883) Google Scholar;Philpott and Paus, 1998Philpott M.P. Paus R. Principles of hair follicle morphogenesis.in: Chuong C.M. Molecular Basis of Epithelial Appendage Morphogenesis. Landes Bioscience Publishers, Austin, TX1998: 75-103Google Scholar). 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, 1992Hardy M.H. The secret life of the hair follicle.Trends Genet. 1992; 8: 55-61Abstract Full Text PDF PubMed Scopus (762) Google Scholar;Philpott and Paus, 1998Philpott M.P. Paus R. Principles of hair follicle morphogenesis.in: Chuong C.M. Molecular Basis of Epithelial Appendage Morphogenesis. Landes Bioscience Publishers, Austin, TX1998: 75-103Google Scholar). Unfortunately, despite a large body of literature on selected aspects of murine HF development, dating back almost a century (Oyama, 1904Oyama R. Entwicklungsgeschichte des Deckhaares der weissen Maus (Mus musculus var. alba).Arb Anat Inst Wiesbaden. 1904; 23: 587-608Google Scholar;Dry, 1926Dry E. The coat of the mouse (Mus musculum).J Genet. 1926; : 287-340Crossref Scopus (230) Google Scholar;Gibbs, 1941Gibbs H.F. A study of the postnatal development of the skin and hair of the mouse.Anat Rec. 1941; 80: 61-81Crossref Scopus (21) Google Scholar;Hardy, 1949Hardy M.H. The development of mouse hair in vitro with some observations on pigmentation.J Anat. 1949; 83: 364-385PubMed Google Scholar;Butcher, 1951Butcher E.O. Development of the pilary system and the replacement of hair in mammals.Ann NY Acad Sci. 1951; 53: 508-515Crossref PubMed Scopus (14) Google Scholar;Chase, 1951Chase H.B. Critical stages of hair development and pigmentation in the mouse.Physiol Zool. 1951; 24: 1-9Crossref PubMed Scopus (113) Google Scholar;Davidson and Hardy, 1952Davidson P. Hardy M.H. The development of mouse vibrissae in vivo and in vitro.J Anat. 1952; 86: 342-356PubMed Google Scholar), there is not a single pragmatic guide that comprehensively and quickly introduces newcomers 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, inHardy, 1992Hardy M.H. The secret life of the hair follicle.Trends Genet. 1992; 8: 55-61Abstract Full Text PDF PubMed Scopus (762) Google Scholar;Paus, 1996Paus R. Control of the hair cycle and hair diseases as cycling disorders.Curr Opin Dermatol. 1996; 3: 248-258Google Scholar;Stenn et al., 1996Stenn K.S. Combates N.J. Eilertsen K.J. Gordon J.S. Pardinas J.R. Parimoo S. Prouty S.M. Hair follicle growth controls.Dermatol Clinics. 1996; 14: 543-558Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar,Stenn et al., 1998Stenn K. Parimoo S. Prouty S. Growth of the hair follicle: A cycling and regenerating biological system.in: Chuong C.M. Molecular Basis of Epithelial Appendage Morphogenesis. Landes Bioscience Publishers, Austin, TX1998: 111-124Google Scholar;Paus & Cotsarchis, 1999Paus R. Cotsarchis G. The biology of hair follicles.N Engl J Med. 1999; 341: 491-497Crossref PubMed Scopus (883) Google Scholar). This comprehensive guide to HF morphogenesis aims at standardizing the presentation of hair research data, and should become a useful tool when screening 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 histochemical 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 slight modifications of previously proposed classification schemes (e.g.,Stöhr, 1903Stöhr P. Entwicklungsgeschichte des menschlichen Wollhaares.Anat H. 1903; 23: 1-66Google Scholar;Hardy, 1949Hardy M.H. The development of mouse hair in vitro with some observations on pigmentation.J Anat. 1949; 83: 364-385PubMed Google Scholar,Hardy, 1951Hardy M.H. The development of pelage hairs and vibrissae from skin in tissue culture.Ann NY Acad Sci. 1951; 53: 546-561Crossref PubMed Scopus (25) Google Scholar;Pinkus and W, 1958Pinkus H. Embryology of hair.in: Montagna W. Ellis R.A. The Biology of the Hair Growth. Academic Press, New York1958: 1-32Crossref Google Scholar;Hardy, 1992Hardy M.H. The secret life of the hair follicle.Trends Genet. 1992; 8: 55-61Abstract Full Text PDF PubMed Scopus (762) Google Scholar). Among the many different HF subtypes (e.g., vibrissae, tylotrich, and non-tylotrich pelage HF) (Sundberg, 1994Sundberg J.P. Handbook of Mouse Mutations with Skin and Hair Abnormalities. CRC Press, Boca Raton1994Google Scholar), 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., 1995Vielkind U. Sebzda M.K. Gibson I.R. Hardy M.H. Dynamics of Merkel cell patterns in developing hair follicles in the dorsal skin of mice, demonstrated by a monoclonal antibody to mouse keratin 8.Acta Anat. 1995; 152: 93-109Crossref PubMed Scopus (53) Google Scholar;Vielkind and Hardy, 1996Vielkind U. Hardy M.H. Changing patterns of cell adhesion molecules during mouse pelage hair follicle development. 2. Follicle morphogenesis in the hair mutants, Tabby and downy.Acta Anat. 1996; 157: 183-194Crossref PubMed Scopus (38) Google Scholar;Paus et al., 1997Paus R. Foitzik K. Welker P. Bulfone-Paus S. Eichmüller S. Transforming growth factor-β receptor type I and type II expression during murine hair follicle development and cycling.J Invest Dermatol. 1997; 109: 518-526Abstract Full Text PDF PubMed Scopus (106) Google Scholar). 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 (Figure 1). Finally, a glossary of anatomical terms frequently used in hair research is also provided so as to avoid terminologic confusion (Table 1).Table 1Glossary of anatomical terms frequently used in hair researchaFor introductory references, see:Stöhr 1903;Oyama 1904;Pinkus 1910;Dry 1926;Hentschel 1930;Hardy 1949,1951;Fleischhauer 1953;Montagna & van Scott 1958;Pinkus 1958;Straile et al. 1961;Parakkal 1969a;Cotsarelis et al. 1990;Holbrook & Minami 1991;Abell 1994;Paus et al. 1994b;Paus & Cotsarchis 1999TermDefinitionBulbProminent, onion-shaped thickening on the proximal end of the HF, consisting of relatively undifferentiated matrix cells, HF melanocytes, as well as of proximal ORS cellsBulbous 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 glandBulge (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 piliDermal papilla (DP)A mesodermal part of the HF, which consists of closely packed mesenchymal cells, framed by the enlarged bulb matrix on the distal endHair canalTube-like connection between epidermal surface and most distal part of the IRS, demarcated by surrounding ORS.Hair coneFirst recognizable part of IRS (pale epithelial layer or Henle’s layer) which starts to develop as a cone-shaped structure above the DPHair 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 dermisHair 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 DPHair shaftThe hair per se, composed of trichocytes (= 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)InfundibulumMost distal part of the HF including hair canal and distal ORS, limited proximally by the duct of SG and laterally by cells of the ORSInner 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.IsthmusSmall part of the HF between insertion of the sebaceous gland and the bulgeOuter 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 epidermisa For introductory references, see:Stöhr, 1903Stöhr P. Entwicklungsgeschichte des menschlichen Wollhaares.Anat H. 1903; 23: 1-66Google Scholar;Oyama, 1904Oyama R. Entwicklungsgeschichte des Deckhaares der weissen Maus (Mus musculus var. alba).Arb Anat Inst Wiesbaden. 1904; 23: 587-608Google Scholar;Pinkus and G, 1910Pinkus F. The development of the integument.in: Kabel G. Mall S. Manual of Human Embryology. Philadelphia, Lippincott1910: 243-291Google Scholar;Dry, 1926Dry E. The coat of the mouse (Mus musculum).J Genet. 1926; : 287-340Crossref Scopus (230) Google Scholar;Hentschel, 1930Hentschel H.G. Die Bildung des Haarpigments nach Untersuchungen an Mäusen.Z Naturw. 1930; 64: 551-596Google Scholar;Hardy, 1949Hardy M.H. The development of mouse hair in vitro with some observations on pigmentation.J Anat. 1949; 83: 364-385PubMed Google Scholar,Hardy, 1951Hardy M.H. The development of pelage hairs and vibrissae from skin in tissue culture.Ann NY Acad Sci. 1951; 53: 546-561Crossref PubMed Scopus (25) Google Scholar;Fleischhauer, 1953Fleischhauer K. Über die Entstehung der Haaranordnung und das Zustandekommen räumlicher Beziehungen zwischen Haaren und Schweissdrüsen.Z Zellforsch Mikroskop Anat. 1953; 38: 328-355Crossref Scopus (4) Google Scholar;Montagna et al., 1958Montagna W. van Scott E.J. The anatomy of the hair follicle.in: Montagna W. Ellis R.A. The Biology of the Hair Growth. Academic Press, New York1958Crossref Google Scholar;Pinkus and W, 1958Pinkus H. Embryology of hair.in: Montagna W. Ellis R.A. The Biology of the Hair Growth. Academic Press, New York1958: 1-32Crossref Google Scholar;Straile et al., 1961Straile W. Chase H. Arsenault C. Growth and differentiation of hair follicles between periods of activity and quiescence.J Exp Zool. 1961; 148: 205-221Crossref PubMed Scopus (93) Google Scholar;Parakkal, 1969aParakkal P.F. The fine structure of anagen hair follicle of the mouse.Adv Biol Skin. 1969: 441-469Google Scholar;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;Holbrook and Minami, 1991Holbrook K.A. Minami S.I. Hair follicle embryogenesis in the human. Characterization of events in vivo and in vitro.Ann N Y Acad Sci. 1991; 642: 167-196Crossref PubMed Scopus (51) Google Scholar;Abell, 1994Abell E. Embryology and anatomy of the hair follicle.in: Olsen E.A. Disorders of Hair Growth. McGraw-Hill, New York1994: 1-20Google Scholar;Paus et al., 1994bPaus R. Handjiski B. Czarnetzki B.M. Eichmüller S. Biologie des Haarfollikels.Hautarzt. 1994; 45: 808-825Crossref PubMed Scopus (17) Google Scholar;Paus & Cotsarchis, 1999Paus R. Cotsarchis G. The biology of hair follicles.N Engl J Med. 1999; 341: 491-497Crossref PubMed Scopus (883) Google Scholar Open table in a new tab 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., 1994cPaus R. Hofmann U. Eichmüller S. Czarnetzki B.M. Distribution and changing density of gamma-delta T cells in murine skin during the induced hair cycle.Br J Dermatol. 1994; 130: 281-289Crossref PubMed Scopus (78) Google Scholar) (Figure 1 and legend for details). Furthermore, we provide a comprehensive guide (Figure 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, 1949Hardy M.H. The development of mouse hair in vitro with some observations on pigmentation.J Anat. 1949; 83: 364-385PubMed Google Scholar,Hardy, 1951Hardy M.H. The development of pelage hairs and vibrissae from skin in tissue culture.Ann NY Acad Sci. 1951; 53: 546-561Crossref PubMed Scopus (25) Google Scholar,Hardy, 1992Hardy M.H. The secret life of the hair follicle.Trends Genet. 1992; 8: 55-61Abstract Full Text PDF PubMed Scopus (762) Google Scholar) (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 1).Figure 2A 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, 1949Hardy M.H. The development of mouse hair in vitro with some observations on pigmentation.J Anat. 1949; 83: 364-385PubMed Google Scholar;Hardy, 1951Hardy M.H. The development of pelage hairs and vibrissae from skin in tissue culture.Ann NY Acad Sci. 1951; 53: 546-561Crossref PubMed Scopus (25) Google Scholar;Hardy, 1992Hardy M.H. The secret life of the hair follicle.Trends Genet. 1992; 8: 55-61Abstract Full Text PDF PubMed Scopus (762) Google Scholar) andPaus et al., 1997Paus R. Foitzik K. Welker P. Bulfone-Paus S. Eichmüller S. Transforming growth factor-β receptor type I and type II expression during murine hair follicle development and cycling.J Invest Dermatol. 1997; 109: 518-526Abstract Full Text PDF PubMed Scopus (106) Google Scholar. 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, 1991Romeis B. Mikroskopische Technik. Urban & Schwarzenberg, München1991Google Scholar). D, G, J, M, P, S, V, Y: AP technique (Handjiski et al., 1994Handjiski B. Eichmüller S. Hofmann U. Czarnetzki B.M. Paus R. Alkaline phosphatase activity and localization during the murine hair cycle.Br J Dermatol. 1994; 131: 303-310Crossref PubMed Scopus (127) Google Scholar) as marker of the configuration and localization of the DP fibroblasts. B, E, H, K, Q, Z: TGF-β RII immunoreactivity (Paus et al., 1997Paus R. Foitzik K. Welker P. Bulfone-Paus S. Eichmüller S. Transforming growth factor-β receptor type I and type II expression during murine hair follicle development and cycling.J Invest Dermatol. 1997; 109: 518-526Abstract Full Text PDF PubMed Scopus (106) Google Scholar). N: IL-1-RI immunoreactivity (Eichmüller et al., 1998Eichmüller S. van der Veen C. Moll I. Hermes B. Hofmann U. Müller-Röver S. Paus R. Clusters of perifollicular macrophages in normal murine skin: physiological degeneration of selected hair follicles by programmed organ deletion.J Histochem Cytochem. 1998; 46: 361-370Crossref PubMed Scopus (73) Google Scholar). 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-RII+ and IL-1-RI+ keratinocytes of the follicular cord and the ORS. T, W: Oil Red O staining (Romeis, 1991Romeis B. Mikroskopische Technik. Urban & Schwarzenberg, München1991Google Scholar) 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-RII+ 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 AP+ mesenchymal cells (d) closely attached to the hair germ (epidermal thickening) (a). (E) the hair germ can only be precisely visualized as TGF-β-RII+ 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) AP+ 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 AP+ 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 keratinocytes (d); the first melanine granules (h) are recognizable above its distal pole. (Q) the TGF-β-RII+ ORS keratinocytes (e) demarcate the elongating TGF-β-RII-negative IRS (a) and the TGF-β-RII-negative DP (d). Stage 6, day 4 p.p. (R) developing sebocytes (a), IRS (c) contains hair shaft (g) with melanin granules; DP (d) is almost completely enclosed and located deeply in the subcutis. (S) hair shaft with melanin granules (g) in the IRS (c), which still had not reached the hair canal (b), AP+ DP (e) deeply located in the subcutis. (T) Oil Red O+ sebum in the hair canal (b), Oil Red O+ SG (f), melanin in the hair shaft (g) still enclosed by the IRS (c). Stage 7, days 4–5 p.p. (U) tip of the hair shaft leaves the epidermis (a) in the close vicinity of the infundibulum of the SG (b). (V) the tip of the hair shaft (a) leaves the TGF-β-RII-negative IRS which is precisely demarcated by the TGF-β-RII+ ORS keratinocytes (c). (W) the tip of the hair shaft (a) in the Oil Red O+ hair canal close to the Oil Red O+ SG (b). Stage 8, days 5–8 p.p. (X) HF at maximal length (a) with a prominent hair shaft (b) emerging through the epidermis. (Y) AP+ DP (d) is located in the deep subcutis. (Z) HF at maximal length (a) revealed by TGF-β-RII+ ORS keratinocytes (c). Scale bars: 50 μm. Please note: upper case letters in the left-hand corner label the image whereas lower case letters identify tissues corresponding to the lower case letters of the criteria listed in the central column.View Large Image Figure ViewerDownload (PPT)Figure 2A 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, 1949Hardy M.H. The development of mouse hair in vitro with some observations on pigmentation.J Anat. 1949; 83: 364-385PubMed Google Scholar;Hardy, 1951Hardy M.H. The development of pelage hairs and vibrissae from skin in tissue culture.Ann NY Acad Sci. 1951; 53: 546-561Crossref PubMed Scopus (25) Google Scholar;Hardy, 1992Hardy M.H. The secret life of the hair follicle.Trends Genet. 1992; 8: 55-61Abstract Full Text PDF PubMed Scopus (762) Google Scholar) andPaus et al., 1997Paus R. Foitzik K. Welker P. Bulfone-Paus S. Eichmüller S. Transforming growth factor-β receptor type I and type II expression during murine hair follicle development and cycling.J Invest Dermatol. 1997; 109: 518-526Abstract Full Text PDF PubMed Scopus (106) Google Scholar. 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, 1991Romeis B. Mikroskopische Technik. Urban & Schwarzenberg, München1991Google Scholar). D, G, J, M, P, S, V, Y: AP technique (Handjiski et al., 1994Handjiski B. Eichmüller S. Hofmann U. Czarnetzki B.M. Paus R. Alkaline phosphatase activity and localization during the murine hair cycle.Br J Dermatol. 1994; 131: 303-310Crossref PubMed Scopus (127) Google Scholar) as marker of the configuration and localization of the DP fibroblasts. B, E, H, K, Q, Z: TGF-β RII immunoreactivity (Paus et al., 1997Paus R. Foitzik K. Welker P. Bulfone-Paus S. Eichmüller S. Transforming growth factor-β receptor type I and type II expression during murine hair follicle development and cycling.J Invest Dermatol. 1997;