MATERIALS AND METHODS

Animals

Female HRS/J hairless (hr/hr) mice, 14–18 wk old, were purchased from Jackson Laboratories (Bar Harbor, ME) and were housed in community cages in the Institute of Toxicology (Free University, Berlin) under standard conditions (12 h light periods, water and mouse chow ad libitum). Only a limited number of untreated, adult HRS/J hr/hr mice were available for analysis from the controls for a study addressing the effects of dioxin on hr mouse skin (^{Panteleyev et al. 1997b}).

Skin sections, histochemistry

The back skin of eight macroscopically hairless hr/hr (–/–) mice was harvested for histology as described elsewhere (^{Paus et al. 1994}). Skin samples were fixed in 4% paraformaldehyde and embedded in paraffin (JUNG-Histowax, Reichert-Jung, Heidelberg, Germany) according to standard procedures. Sections of 5 m were mounted on silane-coated glass slides (six slides with four sections each per mouse) and were prepared for in situ hybridization or were stained with hematoxylin and eosin for routine histology.

Immunohistology

Additional skin samples were frozen in liquid nitrogen immediately after harvesting and were embedded in Tissue-Tek (MILES, Elkhart, IN) medium for storage at –70°C as described (^{Paus et al. 1994}). Air-dried 6 m cryostat sections were collected on silane-coated slides, fixed in cold acetone (–20°C) for 10 min, and then used for histochemical detection of alkaline phosphatase activity (^{Handjiski et al. 1994}) or for immunohistochemistry. For NCAM immunohistochemistry, sections were incubated overnight (room temperature) with primary rat anti-mouse NCAM monoclonal antibodies (Boehringer, Mannheim, Germany) at a dilution of 1:100, and then treated with biotin-SP-conjugated goat anti-rat IgG (Jackson Immunoresearch, West Grove, PA) at a dilution of 1:200. This was followed by incubation with avidin-biotin complex labeled by AP (Vector Laboratories, Burlingame, CA) as described (^{Paus et al. 1994}). Ki-67 immunoreactivity (IR) was studied in paraffin sections (5 m) using rabbit anti-serum (Dianova, Hamburg, Germany) at a dilution of 1:100 (^{Gerdes et al. 1997}), followed by an incubation with biotin-conjugated F(ab)2 fragment of a goat–anti-rabbit IgG (Jackson Immunoresearch; 1:200). Skin sections were then incubated with ABC complex (Vecta-Stain-Kit, Vector Laboratories).

In situ hybridization

In situ hybridization for MK17 expression was performed as described (^{Panteleyev et al. 1997a,b}). For control purposes, the MK17 expression seen in HF of hr mice was compared with the MK17 expression of C57BL/6 mice with normal hair growth patterns (^{Panteleyev et al. 1997a}).

RESULTS

Histologic evaluation

Macroscopically, hairless mouse skin at the age of 3–5 mo is characterized by complete hairlessness. Microscopically, hyperkeratinized utriculi (i.e., epidermis-associated open comedones) that are connected by a short duct to the sebaceous gland (SG) are visible in these mice, together with one or two rows of fully developed DC in the reticular dermis (^{Montagna et al. 1952; Mann 1971}) Figure 1a. Short of being able to sequentially analyze different age groups of hr mice, we wished to distill as many indications to the pathogenesis of the hr phenotype as possible from an analysis of hr skin during this window of postnatal HRS/J hr/hr life.

Figure 1.

Characteristic phenomenology ofhairlessmouse skin (14–18 wk old HRS/J mice) (hematoxylin and eosin). (a) The SG (sg) are connected with the skin surface via the utricular cavity (uc). The utricular epithelium is hyperkeratinized, whereas DC (dc) display a low level of keratinization, as evident from the lack of cornified material in the DC cavities, compared with the utriculi; apm, arrector pili muscle; bc, PBDC. (b) Close-up of the utriculi-SG-unit in hr skin. PBDC clusters at the proximal end of this unit are in intimate association with the arrector pili muscle (apm). They display substantial cellular diversity: the upper portion (up) is constituted of flattened cells with condensed nuclei, which are positioned in one vertical line, forming a cell column. Often, but not always, direct contact between the uppermost cells of these columns and the utricular epithelium (ue) can be appreciated (see Figure 2a). The lower portion (lp) of PBDC clusters consists of relatively undifferentiated cells with clear, swollen cytoplasm, large spherical nuclei, and round contour. (c) A hair-like structure (h) in the deep-located DC (dc). Scale bars, 100 m.

Full figure and legend (41K)

On closer analysis, compact clusters of epithelial cells were noted to be localized just beneath the SG Figure 1b. In these cell clusters, substantial cellular diversity was evident. Their upper portion was surrounded by SG lobules, and consisted of cells with condensed cytoplasm and flattened nuclei, which were positioned in one vertical column, one cell just above the other Figures 1b, 2a. The uppermost cells of these epithelial columns showed direct connections with the epithelium of the utriculi and with the SG duct, but had no other contacts with any epithelial structure Figure 2a. The lower portion of these cell clusters was in direct contact with the insertion of the arrector pili muscle and was composed of cells with clear, swollen cytoplasm, large spherical nuclei, and round contour Figure 1b. This proximal group of epithelial cells sometimes was connected to long downward-oriented strands of epithelial cells Figure 2b, c. In other cases, these strands were replaced by typical DC in different developmental stages (Figure 3a, b, which usually expressed MK17 mRNA (see below; Figure 3c, d.

Figure 2.

Putative bulge cells-associated epithelial strands (bo) in the skin ofhr/hrmice (hematoxylin and eosin). (a) Normal structure of PBDC clusters without signs of associated epithelial strands. Note the direct connection of the utricular epithelium with the upper portion of the PBDC. (b, c) Strands of epithelial cells associated with the lower portion of PBDC clusters. See also Figure 4(e,f). ub, Upper portion of the PBDC; lb, lower portion of the PBDC; uc, utricular cavity; apm, arrector pili muscle; sg, SG; dc, DC. Scale bars, 50 m.

Full figure and legend (106K)

DC in hr skin were well rounded and exhibited a pattern of slow, centripetal differentiation within a thin layer of cyst epithelium Figures 1a, 2a, c, whereas a minority of more proximally located cysts was more unevenly shaped, displayed a thicker cyst epithelium, and contained hair-like filamentous structures Figure 1c.

Alkaline phosphatase activity

In addition to blood vessels and arrector pili muscles, which are positive for AP activity in normal murine skin (^{Handjiski et al. 1994}), positive AP staining in hr skin was detected only in the epithelium of selected DC Figure 4a. Neither regular AP⁺ DP structures, nor AP⁺ DP remnants were detectable. Instead, several AP-positive DC were seen. These were only located deep in the reticular dermis, close to the subcutis (Figure 4a, scm). Interestingly, only AP-positive DC contained hair shaft-like structures Figure 4a,h.

Figure 4.

Histochemical (a, AP activity), immunohistochemical (b–d, NCAM; e–g, Ki-67) staining, andin situhybridization (h, i, MK17 riboprobe) ofhairless mouse skin (14–18 wk oldhr/hrHRS/J mice).Scale bars: a–g, 50 m; h, i, 100 m. (a) AP⁺ DC in the deep dermis. Note the location of these AP⁺ (ap) cysts below the majority of AP DC and near to the subcutaneous muscle layer (scm), the panniculus carnosus. Most of these cysts contain hair fragments (hr), suggesting their origin from abnormal secondary hair folliculoids. (b) NCAM-IR in DC located in the mid-dermis. The NCAM⁺ cells (NC) form a thin sheath around the DC (dc) epithelium, resembling the perifollicular connective tissue sheath of normal hair follicles. (c) NCAM-IR (NC) in DC (dc) containing hair remnants (hr). (d) NCAM-IR in the PBDC in hr mouse skin. The NCAM⁺ longitudinal nerve bundles (nb) surround the upper portion of the PBDC. (e–f) Ki-67 expression on two serial sections of the proximal part of the utriculi-SG unit. Ki-67⁺ cells (Ki) are visible in the utricular epithelium and in the downward-oriented epithelial strand that is associated with PBDC. (g) Ki-67 expression (Ki) in the new DC epithelium, which is formed in close association with PBDC (pbc). (h, i) MK17 mRNA expression (in situ hybridization with a digoxigenin-labeled riboprobe) in the hair follicle infundibulum of C57BL/6 mice with their normal hair coat and cycle (H), and in the utricular epithelium of hairless mice (i). Note the localization of MK17 transcripts to the innermost cell layers of the utricular (ue) and infundibular (inf) epithelium, and the presence of MK17 mRNA in SG ducts (sgd), but not in the SG epithelium in both normal (h) and hr (i) mice. e, Epidermis; dc, DC; sg, SG.

Full figure and legend (379K)

NCAM expression

Unlike normal mouse skin, which is characterized by strong NCAM-IR in the DP and myelinated skin nerves (^{Chuong et al. 1991;Kaplan and Holbrook 1994;Botchkarev et al. 1997;Müller-Röver and Paus 1998}), NCAM-IR in the skin of hr/hr mice, with its absence of normal DP structures, was restricted to a thin layer of connective tissue directly adjacent to the epithelium of selected DC located in the mid-dermis Figure 4b. Very intense NCAM-IR was also seen in the stroma of deep DC that contained hair shaft-like structures Figure 4c and were AP⁺ as well Figure 4a. In addition, the epithelial cell clusters located beneath the SG perform NCAM-IR in their upper portion and were surrounded by strongly NCAM⁺ nerve bundles Figure 4d. The epithelium of utriculi and their adjacent mesenchymal cells, as well as interfollicular dermal fibroblasts or DC located in the upper dermis, displayed no NCAM-IR.

Ki-67 expression

In hr (–/–) skin, prominent Ki-67 IR was found in most basal layer keratinocytes of the utricular epithelium Figure 4e and in the epithelium of most DC (not shown). Contrary to the basal layer of interfollicular epidermis of hr and of normal mouse skin (^{Gerdes et al. 1997}), where only some keratinocytes are Ki-67⁺, nearly all basal keratinocytes in the middle and proximal portion of the utricular epithelium were strongly Ki-67⁺ Figure 4e. This was the case in most follicular remnants, though not in all Figure 4g. Ki-67 IR was also detectable in 10–15% of downward-oriented epithelial strands that were occasionally seen beneath the SG Figure 4e, f, attesting to ongoing cell division in this cell population. In addition, the new DC that formed in the region of the arrector pili muscle attachment also exhibited Ki-67⁺ cells Figure 4g, thus demonstrating high proliferative activity.

MK17 expression

IR denoting MK17 mRNA transcripts was exclusively localized to the epithelial remnants of hr HF. In utricle epithelium, MK17-mRNA expression was detected mainly in the inner cell layers adjacent to the utricular cavity Figure 4i. MK17 staining was seen in the keratinized part of the utricular epithelium as well as in the nonkeratinized proximal part, which corresponds to the SG duct of normal hair follicles. Neither the SG epithelium, nor the more distally located part of the utricles, showed MK17 mRNA expression Figure 4i. The epithelium of the DC expressed MK17 mRNA to a similar degree as the utricle epithelium Figure 4i. The MK17 signal was evenly distributed in the epithelium of all DC. Individual sebocytes that were occasionally visible within the DC epithelium were MK17 negative (not shown).

The compact cell clusters localized proximal to the SG and in association with arrector pili muscle fibers [i.e., putative bulge-derived cells (PBDC)], were normally negative or, at the most, weakly positive for MK17 transcripts; however, whenever these cell clusters were associated with new growing DC Figure 3a, b, their proximal portion exhibited high levels of MK17 mRNA-IR Figure 3c, d.

Hybridization of representative skin sections with MK17 sense-probes as negative controls showed no specific IR (not shown).

DISCUSSION

The origin of utricles and DC in hr mouse skin has so far been uncertain (^{Montagna et al. 1952;Sundberg 1994}). Here, we show that the MK17 expression pattern in utricles Figure 4i resembles the one observed in C57BL/6 mice with their normal hair coat Figure 4h, where the infundibular outer root shealth (ORS) constantly expresses MK17 mRNA (^{Panteleyev et al. 1997a}). This similarity in MK17 expression, and the location of the SG proximal to the utricle epithelium, strongly supports the concept that the utricles in hr skin develop from the infundibular ORS, rather than from other parts of the normal hair follicle epithelium Figure 5.

Figure 5.

Proposed scenario ofhairlessskin pathogenesis. (A) Late catagen (catagen VII according to^{Straile et al. 1961}) in normal hair follicle of C57BL/6 mice. Only the cycling portion (o) of the hair follicle epithelium is regressing, and the DP does not disintegrate. hs, Hair shaft; e, epidermis; sg, SG; b, bulge; irs, inner root sheath; ors, ORS; apm, arrector pili muscle; dp, DP. (B) Late catagen in hr/hr hair follicles. The entire ORS, except for infundibulum and bulge, disintegrates. The ORS disintegration, particularly in the isthmus region, makes the hair shaft, which has been generated during follicle morphogenesis, to loose its mooring in the follicular canal. (C) Hypothetical intermediary stage of the hair follicle disintegration as a consequence of the hr mutation. The ORS is degraded, whereas infundibulum, SG, and bulge cells partially retain their integrity. Clusters of disintegrated central and proximal ORS keratinocytes associate with NCAM⁺ remnants of the former connective tissue sheath (cts). The most proximal portion of these cells (AP⁺ cell clusters) represents remnants of the DP. (D) Structure of adult hairless mouse skin. Utriculi (uc) originate solely from the infundibular portion of the ORS. The bulge-derived cells (b) retain their integrity, but get separated from the proximal follicle epithelium. DC are then formed from three different sources: (i) parts of the disintegrated ORS (NCAM⁺, AP^– stroma); (ii) spontaneous growth activity of PBDC (NCAM^–, AP^– stroma); (iii) AP⁺, NCAM⁺ epithelial remnants of the ORS, possibly in conjunction with DP remnants, which may form epithelial–mesenchymal interaction units that retain the capacity to form incomplete, hair-like structures (h). sg, SG; apm, arrector pili muscle.

Full figure and legend (44K)

The location of the compact clusters of relatively undifferentiated epithelial cells beneath the SG Figure 1a, and their association with arrector pili muscle fibers Figure 1a, b, suggest that these cells correspond to, and derive from, the bulge, which contains the key epithelial stem cells of the normal HF (^{Cotsarelis et al. 1990}). Based on morphologic evidence Figures 2a, b and c, 4e, f, these cells are not a part of the utricular epithelium, from which they are well distinguishable Figure 1b, neither are they "DP-like cells" (^{Sundberg et al. 1991}), because MK17 expression Figure 3c strongly confirms their epithelial nature. It is important to note that the PBDC in hr mouse skin identified here represent an isolated cell conglomerate, whereas bulge cells in normal HF are deeply embedded into a protrusion of the distal ORS (^{Cotsarelis et al. 1990}). This segregation of PBDC from the specific bulge-containing portion of the distal ORS is confirmed by their close association with palisade nerve fibers Figure 4d, which form a specific network around the isthmus region of the normal HF (^{Botchkarev et al. 1997}).

It has previously been proposed that widening of the follicular canal, improper hair club formation, and/or follicular keratosis are the main reasons for hair loss in hr skin (^{David 1934;Fraser 1946;Mann 1971}). The homology of the utricles solely to the infundibular portion of the normal ORS that we demonstrate here Figure 4h, i, and the isolated position of the PBDC in hr mouse skin Figure 1b, suggest not only that the cycling, proximal part of the HF epithelium disintegrates as a result of the hr mutation, but also that the permanent, distal portion of the HF ORS (isthmus) is destroyed Figure 5. Thereby, the hair shafts formed during the course of HF morphogenesis may loosen their mooring in the ORS and fall out, resulting in alopecia. In addition, the DP gets disconnected from the remnants of the HF epithelium (utriculi and bulge-derived cells). This loss of normal epithelial–mesenchymal interactions, in turn, likely disrupts the capacity of HF remnants to cycle.

Though this awaits confirmation, it is reasonable to speculate that this disintegration of the permanent ORS portion results from a dysregulation of the massive, yet tightly controlled keratinocyte apoptosis, which normally occurs in well-defined regions of the follicle epithelium during catagen (^{Weedon and Strutton 1981;Lindner et al. 1997}). We failed to detect an upregulation of TUNEL⁺ cells in the HF remnants of 3–4 mo old hr/hr mice (Panteleyev and Paus, unpublished observation). Because HF degeneration by excessive apoptosis would be expected to occur between the fourteenth and twentieth days of age, and would long have been completed weeks thereafter, this negative result is not at all surprising. The challenge now is to subject hr/hr mice of this age group to TUNEL.

In addition to apoptosis dysregulation, the abnormalities in NCAM expression revealed in hr skin Figure 4c suggest that a collapse of normal HF topobiology, i.e., the shaping and maintenance of follicle morphogenesis and structure by cell adhesion molecules (^{Chuong et al. 1991;Kaplan and Holbrook 1994;Müller-Röver and Paus 1998}), may contribute to HF disintegration in hr skin. Does the hr protein, therefore, represent a transcription factor that normally controls apoptosis and coordinates cell adhesion molecule expression in defined populations of ORS keratinocytes? Interestingly, the remnants of both the HF infundibulum (utriculi) and the PBDC are protected from the – as yet unknown – consequences of lacking the hr gene product, whereas DP- and other ORS-derived cells are not. Still, ORS keratinocytes are not completely extinguished, as evidenced by the formation of various classes of DC by these cells.

The morphologic structure, location Figure 2, and proliferation activity Figure 4e, f of epithelial strands that are in intimate association with the clusters of PBDC are reminiscent of the downward growth of epithelial cells during early anagen of the normal hair cycle (cf.^{Paus 1996;Stenn et al. 1996}). Therefore, the downward-oriented epithelial strands in hr skin may represent outgrowths of the PBDC.

We show that the PBDC in hr skin also have the capacity to produce DC Figures 3a, b, c and d, 4g. These cysts are quite similar to trichoepitheliomas, which are thought to be produced by an abnormal proliferation of bulge cells (^{Pinkus 1951}). Therefore, hr skin might offer a useful model for studying the mechanisms of trichoepithelioma induction.

Which signals may induce the growth of cells in the PBDC of hr mouse skin? In normal HF, the role of the DP in the generation of such a signal is widely accepted (^{Jahoda et al. 1984;Cotsarelis et al. 1990;Paus 1996;Stenn et al. 1996}). Because AP activity and NCAM-IR are characteristic markers for normal DP fibroblasts (^{Chuong et al. 1991;Handjiski et al. 1994;Kaplan and Holbrook 1994;Combates et al. 1997}), the absence of AP⁺ and NCAM⁺ cells in the proximity of the PBDC in hr skin suggests that DP-derived signals are not essential for inducing the outgrowth of these cells. This raises the possibility that cyclic growth activity is generated from within this particular epithelial cell population, independent of inductive signals from a specialized mesenchyme. The lack of appropriate survival signals from a specialized mesenchyme (^{Raff et al. 1993}) in hr skin, however, may be a limiting factor for this proposed, autoinduced bulge cell proliferation.

Although regular DP structures are absent in the skin of hairless mice, we here describe ectopic AP activity associated with NCAM-IR in the stroma of selected DC in the reticular dermis Figure 4a, c. The observation that only these AP⁺ and NCAM⁺ DC contain hair-like structures Figure 1c suggests that they originate from HF remnants that retain (at least in part) the ability to produce incomplete hair shafts, perhaps due to the presence of morphogen-secreting AP⁺ and NCAM⁺ remnants of disintegrated DP fibroblast populations Figure 5.

The origin of DC in the skin of hr mice is a subject of much debate. Formerly, it had been suggested that they originate, at least in part, from SG (^{Crew and Mirskaia 1931;Montagna et al. 1952}); however, several lines of evidence strongly suggest a nonsebaceous origin of DC in hr skin: DC are present even in the skin of double-homozygous hairless and asebia mice (hr/ hr, ab/ ab) that are characterized by a complete absence of SG (^{Gates et al. 1969}). Also, the NCAM⁺ sheath of DC in hr skin Figure 4b resembles the NCAM expression of the perifollicular connective tissue sheath of normal HF (^{Chuong et al. 1991}), supporting that DC derive from the ORS. Finally, MK17 mRNA expression was detected in the epithelium of all DC Figure 4i, but never in SG, neither in hairless skin Figure 4i nor in normal C57BL/6 mice Figure 4h (^{Panteleyev et al. 1997a}), nor in humans.¹

Because murine HF with normal growth and cycling express MK17 in the innermost cell layer of the proximal ORS (^{Panteleyev et al. 1997a}) that is normally surrounded by a NCAM⁺ connective tissue sheath (^{Chuong et al. 1991}), the MK17⁺ DC epithelium of hr mice surrounded by thin NCAM⁺ connective tissue sheath most likely arises from remnants of the disintegrating, proximal ORS (cf.^{Bernerd et al. 1996}). This is further supported by the finding that clusters of proximal ORS keratinocytes from human anagen HF cultured under three-dimensional conditions can form spheroid structures with centripetal differentiation (^{Limat et al. 1994}), not unlike the DC of hr mice.

Taken together, this suggests three possible pathways of DC morphogenesis in the skin of hr mice Figure 5: (i) from the disintegrating ORS, resulting in cysts positioned in the central dermis that display an NCAM⁺ connective tissue sheath; (ii) from PBDC, resulting in AP^–, NCAM^– cysts positioned in the upper dermis; (iii) from as yet ill-characterized epithelial structures that are located deep in the dermis (perhaps in conjunction with surviving AP⁺ and NCAM⁺ DP fibroblasts), and that retain the capacity to form incomplete hair-like structures. These different pathways of DC morphogenesis would help to explain the long-appreciated pluripotency of the DC epithelium in hr mice, which is capable of lipogenesis, squamous differentiation (^{Montagna et al. 1952;Sundberg 1994}), and the formation of hair-like structures, corresponding to the distinct pathways of epithelial differentiation seen in normal HF (^{Pinkus et al. 1981}).

Thus we propose that one of the main cellular mechanisms of hair loss in hairless mice, and the main reason for the subsequent failure of hair follicle cycling, is the disintegration of most of the upper ORS due to loss of the hr gene product Figure 5, a putative transcription factor (^{Cachon-Gonzalez et al. 1994}). This may cause profound alterations in the transcription of genes involved in the control of ORS keratinocyte apoptosis and of follicle topobiology (cf.^{Lindner et al. 1997;Müller-Röver and Paus 1998}).

The improved understanding of the histogenesis of the hairless phenotype that this study provides, invites one to exploit the hr mutation as a unique model for dissecting the interactions of bulge and DP as well as the transcriptional control of HF regression under physiologic and pathologic conditions. Given that premature or retarded HF regression is pivotal to the development of most common hair growth disorders seen in man (^{Paus 1996}), analysis of the molecular and cellular pathology of the hr mutation may provide critical pointers to the elusive key genes of catagen control (cf.^{Paus 1996;Stenn et al. 1996}), and to more efficient means for manipulating their expression during the treatment of human hair growth disorders.

Notes

¹ Holland DB, Roberts SG, Cunliffe WJ: Localization of keratin 17 mRNA in acne. J Invest Dermatol 108:384, 1997 (abstr.)

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Acknowledgments

We are most grateful to Dr. J. Schweizer (German Cancer Research Center, DKFZ, Heidelberg) for supplying us with the MK17 cDNA clone, to Dr. R. Wanner for help with the riboprobe preparation, to Dr. R. Thiel for valuable assistance with housing hr mice and with skin collection, to Dr. V.A. Botchkarev and to B. Handjiski for constructive criticism, and to Prof. W. Sterry for advice and encouragement. This work was supported in part by an ESF Research Fellowships in Toxicology to A.A.P., by a grant from the German Ministry for Research and Technology (BMFT 07 DIX 15) to T.R., and by grants from Deutsche Forschungsgemeinschaft (Pa 345/3–2) and Wella AG, Darmstadt, to R.P.