Smooth muscle a-actin is a marker for follicle in vivo and in vitro

COLIN A. B. JAHODA1*, AMANDA J. REYNOLDS2•*, CHRISTINE CHAPONNIER2, JAMES C. FORESTER3 and GIULIO GABBIANI*

1Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, Scotland ^Department of Pathology, University of Geneva, 1211 Geneva 4, Switzerland ^Department of Surgery, Ninewells Hospital and Medical School, Dundee, Scotland * Present address: Department of Biological Sciences, University of Durham, Durham DH1 3LE, England

Summary

We have examined the expression of smooth muscle cells contained significant quantities of the a-actin a-actin in hair follicles in situ, and in isoform. dermal cells in culture by means of immunohisto- The rapid switching on of smooth muscle a-actin chemistry. Smooth muscle a-actin was present in the expression by dermal papilla cells in early culture, dermal sheath component of rat vibrissa, rat pelage contrasts with the behaviour of smooth muscle cells and human follicles. Dermal papilla cells within all in vitro, and has implications for control of ex- types of follicles did not express the antigen. How- pression of the antigen in normal adult systems. The ever, in culture a large percentage of both hair very high percentage of positively marked cultured dermal papilla and dermal sheath cells were stained papilla and sheath cells also provides a novel marker by this antibody. The same cells were negative when of cells from follicle dermis, and reinforces the idea tested with an antibody to desmin. Overall, explant- that they represent a specialized cell population, derived skin fibroblasts had relatively low numbers contributing to the heterogeneity of fibroblast cell of positively marked cells, but those from skin types in the skin dermis, and possibly acting as a regions of high hair-follicle density displayed more source of myofibroblasts during wound healing. smooth muscle a-actin expression than nbroblasts from areas with fewer follicles. 2-D SDS-PAGE Key words: smooth muscle or-actin, hair follicle, nbroblasts, confirmed that, unlike nbroblasts, cultured papilla dermal heterogeneity.

Introduction dermal papillae after papilla removal, or lower follicle amputation (Oliver, 19666; Jahoda et al. 1991). Skin appendage development is engineered through a The finding that cultured rat vibrissa dermal papilla series of interactions involving dermal mesenchyme and cells (Jahoda and Oliver, 1981) retain their capacity to epithelial-derived (Sengel, 1986, for review). In stimulate hair growth (Jahoda et al. 1984; Home et al. hair follicle formation, the mesenchymal cells that form 1986) distinguishes them as a culture system with the dermal component of the structure first become visible particular relevance to known biological function. Other as a cell agglomeration just below a primary epidermal investigations have used cultured dermal components thickening. At an early stage, cell division within the from human hair follicles (Messenger, 1989; Arai et al. mesenchymal aggregation apparently ceases (Wessels and 1989 for reviews). The morphology, behaviour and biosyn- Roessner, 1965), and these cells form the dermal papilla thetic activites of papilla cells reflect their distinct in situ component during follicular downgrowth. Connected with properties (Jahoda and Oliver, 1984; Messenger et al. 1986; the base of the adult dermal papilla, and separated from Couchman, 1986), and it has been suggested that papilla the outermost epidermal layer of the follicle by a thick cells represent a specialized skin fibroblast population , is the external element of the follicle (Jahoda and Oliver, 1984). This view is supported by the mesenchyme, termed the dermal sheath. finding that fibroblasts derived from skin explant cultures Microsurgical manipulation of the rat vibrissa follicle are incapable of hair growth stimulation (Jahoda et al. model system has shown that the adult dermal papilla is a 1984; Home et al. 1986). However, while certain features prerequisite for hair growth initiation and maintenance such as the synthesis of basement membrane extracellular (Oliver, 1966a,6,1967). Moreover, the adult dermal papilla components (Couchman, 1986) help to distinguish papilla retains powerful inductive capabilities in that it induces cells from skin fibroblasts, none can be expressly described new hair follicle formation when associated with afollicu- as a specific papilla cell marker. lar or wound epidermis (Oliver, 1970; Reynolds, 1989). The This question fringes on the broader problem of vibrissa follicle dermal sheath cells also possess distinc- categorization of the cells that are present in the skin tive properties, since they can be the source of regenerated dermis, and which commonly appear fibroblast-like when Journal of Cell Science 99, 627-636 (1991) Printed in Great Britain © The Company of Biologists Limited 1991 627 put into culture. It has become increasingly apparent that muscle cell cytoskeletal characteristics, an idea previously dermal subpopulations exist within adult skin, and that mentioned by Couchman (1986). In the current study we explant culture in particular may produce heterogeneous used immunolabelling methods to examine human and rat fibroblast outgrowth for investigative purposes. One group follicular elements in vivo and in vitro, and report that the has recently attempted to tackle this problem by adopting two principal dermal components, the dermal papilla and a classification system that separates fibroblasts into a the dermal sheath, both express vascular smooth muscle a series of differentiation states according to their morpho- actin in vitro. logical and behavioural properties (Bayreuther et al. 1988). A distinctive fibroblastic cell type first described in Materials and methods granulation tissue, and associated with retractive pro- Rat tissues were derived from an inbred strain of PVG rats cesses of soft tissues, is the myofibroblast. These cells (Colony Dundee University), and from Wistar rats. Human possess a microfilamentous system akin to that observed material came from groin, scalp and scrotal skin biopsies as a in cultured fibroblasts or smooth muscle cells. More result of surgical procedures. recently, myofibroblasts have been described in normal Pieces of rat skin were taken from body regions with high hair tissues, where it has been proposed that they exert follicle density (mystacial pad), reduced hair follicle density (ear contractile activities (Sappino et al. 1990). The use of skin), and without follicles (footpad skin). Isolated vibrissa cytoskeletal markers such as desmin, an intermediate follicles were dissected from the mystacial pad region as filament protein typical of most muscle cells; smooth previously described (Jahoda and Oliver, 1981), and human muscle ir-actin, an actin isoform found in smooth muscle follicles were dissected from groin, scalp and scrotal skin with watchmakers' forceps and curved iridectomy scissors. cells; and smooth muscle myosin, has enabled several For tissue immunohistochemistry, specimens were embedded myofibroblastic subpopulations in normal and pathologi- in Tissue tek HI (Miles Scientific) water-based mounting medium cal tissues to be defined. Using a monocolonal antibody and snap frozen over liquid nitrogen. Cryostat sections of 6 /on that specifically recognizes smooth muscle o--actin Skalli et were cut at -20°C and air dried prior to labelling. al. (1986) noted that some hair follicle dermis stained positively. This, and resemblences between cultured Cell cultures dermal papilla cells and myofibroblasts prompted us to Dermal papilla cultures were established from intact papilla investigate whether hair follicle mesenchyme had smooth- explants (Fig. 1) dissected from vibrissa, and human follicles, as

Fig. 1. An isolated vibrissa dermal papilla prior to being put into culture. Note how papillae can be cleanly separated from the follicle epidermis, and the dermal sheath. Phase-contrast; x360. Fig. 2. A human dermal papilla still attached to dermal sheath (ds) that has been inverted during the dissection procedure. The two are then separated at the narrow basal stalk region (arrowed). Phase-contrast; X170. Fig. 3. Dermal papilla cell culture treated with pre-immune serum in the staining procedure as one of the immunohistochemical controls.

628 C. A. B. Jahoda et al. previously described (Jahoda and Oliver, 1981; Messenger, 1984). buffer (pH7.2) and 0.05% Ruthenium Red (to enhance visualiz- Vibrissa dermal sheath cultures were initiated from the thin ation of extracellular material) for two hours. Postfixation was in circular envelope that remains attached to the dermal papilla 1% osmium tetroxide, with 0.1M cacodylate buffer and 0.025% after removal of the epidermal component during dissection. Ruthenium Red, for three hours at 4°C. Material was dehydrated Human dermal sheath cells were cultured from all of the bulbar in increasing concentrations of ethanol and propylene oxide dermis remaining after the papilla and epidermal matrix has before being embedded in Epon resin. Ultrathin sections were been excised (Jahoda et al. unpublished). Briefly, the bases of stained with uranyl acetate, followed by lead citrate, and anagen follicles were cleared of any adherent tissue, under a examined with a Zeiss EM 109 electron microscope. binocular dissecting microscope (x20) in Minimal Essential Medium containing penicillin/streptomycin at 50 units ml"1 (both Gibco), and their bulb regions were excised and transferred to dishes with fresh medium. The follicle bulbs were then inverted using a combination of finely tipped watchmakers' forceps and sharp tungsten needles. This invariably led to separation of the Results epidermal component from the dermal papilla and attached sheath of follicular dermis (Fig. 2). The sheath was then easily Staining of normal tissue sections separated from the papilla with tungsten needles. Rat vibrissa follicles. In anagen vibrissa follicles, Rat skin fibroblast cultures came from explants from the three overall actin distribution, as revealed by the general above mentioned regions of skin: mystacial pad, ear and footpad. antibody, was widespread, with the outer of Where possible, fibroblast cultures were set up from the same the follicle epithelium strongly marked (Fig. 4). individuals as were used to provide papilla and dermal sheath Smooth muscle u-actin labelling of mid-anagen vibrissa cells. Areas of skin were cleaned, and surface hair fibres removed, follicles revealed that the dermal papilla component was before the tissue was chopped into small pieces. Fibroblast virtually unmarked. However, the lower dermal sheath of cultures were set up from explants of in a similar manner. the follicle was clearly labelled, except for a region For immunolabelling of cells during early outgrowth, explants surrounding part of the bulb at the base of the follicle were established directly on top of glass coverslips in 35 mm (Fig. 5). This marking clearly delineated a line of dermal culture dishes. For later observation, passaged cells were seeded sheath cells just exterior to the glassy membrane (Fig. 5) at various densities on top of coverslips in 35 mm dishes. All but from about half way up the follicle, staining became cultures were fixed and permeabilized by exposure to methanol patchy, and then petered out (Fig. 6). Follicles were not for 30min at — 20 "C before marking. labelled by the antibody to desmin but vibrissa sections acted as an excellent positive control for this antibody, Immunohistochemistry because follicles could be left with some of their surround- Cultured cells and sections were incubated with a monoclonal ing muscle tissue attached, and these muscle blocks antibody to smooth muscle o^actin (anti-asm-1, Skalli et al. 1986) always displayed intense staining (Fig. 7). diluted 1:10 in phosphate-buffered saline (PBS), pH7.4, at room Rat skin. Foot pad skin was virtually unmarked, with temperature for one hour. After thorough washing in PBS, only dermal vasculature and Meissner corpuscles posi- material was exposed to biotinylated anti-mouse antibody (BRL), tively labelled. Ear and whisker pad skin both showed diluted 1:90, for one hour, washed again, and incubated with fluorescein-linked streptavidin, diluted 1:100, for the same length widespread follicular staining with the total actin anti- of time. Specimens were mounted in Citifluor (Agar aids) body (Fig. 8). As in vibrissa follicles, smooth muscle a- medium, and examined and photographed on a Zeiss ICM 405 actin label was restricted to the dermal sheath, with no inverted microscope equipped with epifluorescence. Alterna- expression at all inside the dermal papilla except for tively, for more permanent preparation the material was stained occasional marking of blood vessels (Fig. 9). However, the as described above, but with horseradish peroxidase-linked smaller pelage follicles differed from vibrissae in that steptavidin. Estimations of positive smooth muscle actin marking labelling extended right around the follicle base. There in cell cultures, were carried out by examinining one hundred was also an indication that staining of the dermal sheath cells in randomly chosen fields. Rat tissues and cell cultures were was absent or reduced in follicles that had shortened, and also labelled with a polyclonal antibody that recognizes all actin were in the telogen phase of the hair cycle. Intriguingly, isoforms (Skalli et al. 1986). The same material was tested for desmin with an affinity-purified rabbit anti-desmin polyclonal longitudinal sections cut at the outside edge of follicles antibody (Kocher et al. 1984) and a monoclonal antibody to revealed a highly organized pattern of smooth muscle a- vimentin (Transformation Research, Inc. Framingham, MA, actin within the dermal sheath. The label showed up as a USA). As one of the control procedures, specimens were treated stack of concentric horizontal rings around the follicle with pre-immune sera. None of the control sections or cultures circumference (Fig. 10). This tied in with parallel obser- treated in this fashion showed any significant fluorescence vations from median sections where the outer line of (Fig. 3). dermal sheath fluorescence was punctuated rather than continuous. Gel electrophoresis Human follicles. Anagen human hair follicles were also For 2-D PAGE, passage-two cultured dermal papilla cells and marked around the dermal sheath and, like rat vibrissa fibroblasts were dissolved in sample buffer containing 1 % sodium ff follicles, label was not always strong outside the lowest dodecyl sulphate, 1% dithiothreitol, limn N -p-tosyl-L-arginine part of the follicle bulb. In some specimens the basal stalk methyl ester and lmM phenylmethylsulphonyl fluoride. In the below the papilla was strongly marked (Fig. 11), but first dimension, actin isoforms were separated by isoelectric focusing according to OTarrell (1975). The pH gradient was papillae were never stained. Unlike vibrissa follicles, established with 2% preblended Ampholines. pH 4.0-6.5 (LKB dermal sheath labelling extended into the upper half of Co., Lucerne, Switzerland). The second dimension was run on appendages. Detailed exmination showed that the anti- SDS-10% PAGE. For the different cell types, equal amounts of body was only staining the internal cell layer of the sample protein were loaded, following protein determination by dermal sheath. When this was cut tangentially, it showed the Bradford (1976) method. the same circular horizontal labelling observed in the dermal sheath of rat pelage follicles. The outer connective Electron microscopy tissue sheath remained unmarked, although blood vessels Specimens were fixed in 2% glutaraldehyde in 0.1M cacodylate peripheral to the follicle were often fluorescent (Fig. 12).

Smooth muscle a-actin in hair follicle dermis 629 Passaged cell culture labelling cultures. Indeed, several dermal sheath cell cultures Smooth muscle a-actin. In early-passage rat dermal appeared to have nearly every cell stained positively, cells (passages 1 to 3), hair follicle-derived dermal papilla although within groups individual cells displayed differ- and dermal sheath cells were both highly positive for ent intensities of label. The distribution of marker within smooth muscle o--actin; inside prominent stress fibres cells reflected previous observations, in that marking of (Figs 13, 14). Although the amount of positive staining stress fibres did not always extend to the periphery of the was slightly variable between different lines, it was cell projections. However, intricate patterns of cyto- estimated that over 75 % of papilla cells, and more than skeletal architecture were visible near the periphery of 95% of dermal sheath cells, were marked in all these many larger flattened cells, perhaps associated with

630 C. A. B. Jahoda et al. Figs 4-12. In situ labelling. Explant outgrowth labelling Fig. 4. Longitudinal section through a rat vibrissa follicle Vibrissa dermal papilla and dermal sheath. Cells showing total actin expression. Within the follicle, the most around fragments of dermal sheath tissue were univer- strongly marked elements are the epidermal sally marked by the smooth muscle o--actin antibody as (arrowed) and the lower dermal sheath, just outside the soon as they emerged from the explants. By contrast, cells dermal-epidermal junction. x55. Fig. 5. The same follicle labelled with smooth muscle oactin. from dermal papilla explants that had attached and Apart from blood vessels outside of appendage, only the lower flattened were initially negative. Then, between the first dermal sheath is stained, by a discrete line of marking. This and second week in culture as cells began to move and label does not extend proximally around the base of the bulb, spread out, their appearance altered. Cells at the becomes increasingly fragmented and then disappears, above periphery of the explants became positive for the smooth the lower third of the structure. The dermal papilla is totally muscle a-actin antibody, while the central mass still unmarked. X55. remained negative (Figs 18, 19). In papilla explant cul- Fig. 6. Detail of dermal sheath marking shows that tures that had been established for 14 days or more, when fluorescence is located just outside the glassy membrane. The cells had further dispersed, an increasingly high pro- line of label is seen to be punctuated rather than continuous, portion of them were marked. and becomes more diffuse and fades distally. x220. Fig. 7. Vibrissa follicle stained with antibody to desmin. Mystacial pad skin. In the cell-covered substratum Within the follicle neither the dermal papilla (p) nor the surrounding pieces of mystacial pad skin, whole regions dermal sheath is marked, but the muscle blocks outside the were completely unmarked, and it was clear that positive follicle capsule are strongly labelled. xllO. staining among the fibroblast cells was highly localized. Fig. 8. Pelage follicles in longitudinal section stained with Further scrutiny of areas with labelled cells revealed that antibody to total actin. Marking is clearest in the outer root label was usually found around pelage hair follicles. sheath of the epidermis, and the dermal sheath, x 120. Follicles were visible in the skin explants and groups of Fig. 9. The same region of skin stained with smooth muscle marked cells were seen around the outline of these a<-actin. As with vibrissa follicles, label is restricted to the follicular 'contaminants' (Fig. 20). dermal sheath; however, in the case of anagen pelage follicles staining extends right around the follicle bulb proximally. The Human cells. Between passages 1 and 3, widespread dermal papilla is not labelled. XllO. staining of the dermal papilla and dermal sheath cells Fig. 10. Closer view of the dermal sheath region of a pelage with smooth muscle

Smooth muscle a-actin in hair follicle dermis 631 displayed extensive cytoplasmic processes, many of which These cells were undoubtedly in the process of moving were packed with microfilaments. Sometimes, elongated (Figs 27, 28), and appeared most often during periods of cells were observed with bulbous regions of cytoplasm changing activity, at the beginning or end of the hair bridged by narrower sections rich in microfilaments. cycle.

Figs 13-17. Staining of passaged rat cells. Fig. 13. Passage 3 rat dermal papilla cells, many of which are overlapping, show widespread labelling with smooth muscle oactin. x90. Fig. 14. Rat dermal sheath cells after three passages display strong marking with the smooth muscle a-actin antibody. Note the broad flattened morphology of this cell type. x90. Fig. 15. One a-actin-labelled cell against a field of unstained cells in a passage 2 ear skin fibroblast culture. x56. Fig. 16. Antibody to total actin marks stress fibres in every cell in this passage 2 skin fibroblast culture. X220. Fig. 17. Vimentin filaments labelled in a passage 2 dermal papilla cell. x380.

Fig. 18. A dermal papilla explant after 11 days. The centre of second passage, marked with smooth muscle a^actin. Note the the explant (e) has collapsed, and papilla cells have attached to relatively broad morphology of some cells. x90. the substratum but are still relatively small and tightly Fig. 22. A second-passage groin skin fibroblast culture, bunched. There is almost no smooth muscle o^actin staining in showing small numbers of labelled cells, against an unstained this central area but some cells are marked towards the edge background of confluent cells. Many parts of human skin of the field. X90. fibroblast cultures were completely unmarked. x90. Fig. 19. Lower magnification view of the same explant culture. Fig. 23. Groin dermal sheath cells positively stained for a- Cells at the core of the explant are unmarked, but towards the actin streaming outwards from a central tissue explant, which periphery, where they have become more spaced out and is itself well marked. Ten day culture. x90. flattened, there is widespread marking. x56. Fig. 24. A human groin dermal papilla explant (p) equivalent Fig. 20. Low magnification view, showing two hair follicles at to that in Fig. 23, is not positively labelled by the tr-actin the edge of a skin explant, and two clumps of smooth muscle antibody at ten days. Of the cells that have emerged and settled cr-actin-labelled cells in close proximity (arrowed). In fibroblast in the vicinity, a relatively small proportion are marked. x225. explant culture, the only large groups of positively stained Fig. 25. (A),(B) Two-dimensional polyacrylamide gel cells were invariably associated with hair follicles, and were electrophoresis (2-D PAGE) of second-passage cultured cells. usually in aggregated form. X56. Dermal papilla cells contain a<-, /?- and y-actins (A), wheras Fig. 21. Human groin follicle dermal papilla cells after their skin fibroblasts only show /S- and ^actin isoforms (B).

632 C. A. B. Jahoda et al. Discussion epithelial interactions that are known to govern organo- genesis. The recognition of heterogeneity in the cyto- It appears increasingly clear that the cells that are skeletal differentiation programme of fibroblastic cells collectively called fibroblasts or stromal cells display a may therefore help us to understand further the special- phenotypic diversity that was not previously suspected on ized activities of the different subpopulations. Cytoskel- morphological grounds (Sappino et al. 1990). As the etal markers are useful for this purpose, and are predominant cell type in connective tissue, the fibroblast particularly suited to identifying potential contractile fulfils a variety of essential functions, and plays a pivotal capacities of fibroblastic cells. role in tissue repair. It is also implicated in mesenchymal- As briefly observed previously (Skalli et al. 1986), the

Smooth muscle a-actin in hair follicle dermis 633 Fig. 26. Transmission electron microscopy of a cultured dermal papilla cell sitting on top of another within a typical papilla cell aggregate. A prominent microfilament band (arrowed) lies at the base of this cell, x 16 000. Fig. 27. Transmission electron microscopy of a vibrissa follicle dermal papilla at catagen. One papilla cell (arrowed) is highly elongated, and is apparently in the process of moving position. X4000. Fig. 28. Higher magnification view of the microfilament structure (arrowed) in the same cell. X22000. dermal sheath region of the hair follicle labels positively The change of papilla cells from negatively stained in with smooth muscle a'-actin, and present work shows that vivo to positively marked in vitro has a parallel in cultured in vibrissa follicles dermal sheath marking is often eye lens cells (Schmitt-Graff et al. 1990), and it dis- restricted to the lower half of the follicle. This observation tinguishes these special types of fibroblastic cells from may relate to experimental work where, on removal of the adult smooth muscle cells whose smooth muscle a'-actin lower third of vibrissa follicles, a new papilla is re-formed content decreases when placed in culture. During athero- from lower dermal sheath cells. The latter are observed by sclerosis, smooth muscle cells also lose smooth muscle a'- light and electron microscopy migrating from the sheath actin and revert to having a /3-isoform predominance, region through the thick specialized basement membrane similar to that seen in embryonic tissue (Gabbiani et al. of the follicle to the site of papilla formation (Oliver, 1984; Kocher and Gabbiani, 1987). While dermal sheath 19666: Jahoda et al. 1991). When the follicle is ablated cells did not alter their smooth muscle a--actin expression higher up, papilla regeneration does not take place in culture, a feature of the current work was the very rapid (Oliver, 1966a). While sheath cells can definitely alter switch on of smooth muscle o--actin expression by papilla their phenotype to become papilla cells, it is less clear cells around the second week of explant culture. The whether the reverse is true, although characteristic finding that cells that moved into spaces away from the Alcian Blue marking of papilla cells suggests that they at original papilla mass immmediately started to express least partially assume the role of dermal sheath in new smooth muscle o--actin, and that the whole culture could follicles induced by papilla interaction with skin epider- transform in a matter of days, depending on the speed of mis (Oliver, 1970). Human follicle papilla regeneration dispersal, was intriguing. It suggests that perhaps the can also be elicited (Jahoda et al. 1989), which suggests papilla cells are held in a state of non-expression by that a similar papilla/sheath cell relationship exists in the modulatory or inhibitory factors within the follicle, which lower human follicle. The finding that only the internal, are lost in the culture environment; this is unusual in horizontally aligned layer of the human dermal sheath relation to control of smooth muscle differentiation. On has smooth muscle a'-actin marking, emphasizes that this this point it is worth noting that the dermal papilla has a structure essentially consists of two layers (Kligman, glycosaminoglycan (GAG)-rich extracellular matrix, and 1988), each of which may have a separate function. This that heparin is one extracellular component that is known may also hold true for the rodent pelage follicle sheath, to influence smooth muscle a^actin expression in vivo and indeed for other follicle types. (Clowes et al. 1988), and in vitro (Desmouliere et al. 1990). The sudden change in marking also provided a possible Here we report for the first time that cultured cells from link between smooth muscle a<-actin expression and cell both the dermal papilla and the dermal sheath are marked motility, since papilla cells took up this label at the time positively with smooth mucle o--actin-specific antibodies, when they began to move. In this context, ultrastructural but react negatively with desmin antibody. Like skin observations of migratory papilla cells in situ stress the fibroblasts, both sheath and papilla cells contained need to consider the papilla cell population as dynamic and vimentin. While counts of numbers of smooth muscle a'- not static, particularly during periods of morphogenetic actin positive cells acted as a useful indicator, they could activity. not be regarded as accurate assessments of overall content because of individual variation in labelling intensity. Another striking finding was the relative homogeneity However, 2-D PAGE analysis confirmed that the smooth of the dermal sheath cell cultures, where an exceptionally muscle a'-actin isoform is a major component in the high proportion of cells stained positively with smooth cytoskeletal make up of follicle dermal cells, but not an muscle a'-actin antibody. This means that dermal sheath appreciable element in skin fibroblasts. could become a valuable source of culture material for

634 C. A. B. Jahoda et al. studies of smooth muscle a--actin as a cell cytoskeletal smooth muscle characteristics of dermal sheath cells could component. relate to contractile processes that might control hair In relation to papilla cell cultures, the reduction in follicle shortening and upward fibre movement during the numbers of positively stained cells over time could be a hair growth cycle. In this context, the observed alignment reflection of the ageing of the cell lines. It has previously of smooth muscle a^actin in the inner follicle dermal been observed that human papilla cells have a relatively sheath as a series of concentric circular rings clearly short lifespan before they become 'senescent' (Messenger, implies functional importance, particularly as this organ- 1989). The low numbers of marked cells seen in a passage 7 ization appears to be common to many follicle types. human papilla cell culture coincided with a slowing down This study provides a new marker for hair follicle in cell division and other signs of senescence at the time of dermis, and possibly an opportunity of examining the hair testing. follicle from a slightly different evolutionary and develop- Although rat skin fibroblast cultures generally had low mental point of view. It will certainly assist in those numbers of cells containing smooth muscle a--actin, those investigations where hair follicle cells need to be dis- from hairy skin (whisker pad) appeared to have more tinguished. labelled cells than cultures from less hairy regions (footpad or ear). This tied in with the observation that C.A.B.J. held a Royal Society, 1983 University Research early on in skin explant culture positively stained cells Fellowship. This work was supported in part by the Swiss had a patchy distribution, and were seen grouped at the National Science Foundation, grant nos 3.108- 0.88 and 31- periphery of hair follicles. In rat fibroblast cell lines we 26614.89, and the Wellcome Trust. have observed heterogeneity of morphology, and from time to time have noted aggregations of cells, similar to those observed in dermal papilla (Jahoda and Oliver, 1984) or References dermal sheath (Jahoda et al. unpublished) cultures. From ARAI, A , KATSUOKA, K., KIESWETTER, F., SCHELL, H. AND HORNSTEIN, O. these observations, one idea is that fibroblast cell lines P. (1989). Effects of testosterone, dihydrotestosterone and estradiol on from skin explants that incorporate hair follicles are the growth behavior of cultured hair bulb papilla cells and root 'contaminated' by follicular mesenchyme cells. This could aheath flbroblaste. In Trends m Human Hair Growth and Alopecia Research (ed. D. Van Neste, J. M. Lachapelle and J. L. Antoine), pp. have significance with regard to fibroblast lineage re- 99-104. London: Kluwer. lationships. It also might need to be taken into consider- BAYREUTHER, K., RODKMANN, H. P., FRANCZ, P. I. AND MAIER, K. (1988). ation in experimental work on skin fibroblasts, where the Differentiation of fibroblast stem cells. In Stem Cells (ed. B. I. Lord original tissue source is hair-bearing skin. However, the and T. M. Dexter), J. Cell Sci. Suppl. vol 10, pp. 115-130. Cambridge: finding of the same phenomenon in human explants raises The Company of Biologists Ltd. BRADFORD, M. M. (1976). A rapid and sensitive method for the a potentially more important idea. Myofibroblasts that are quantification of microgram quantities of protein utilizing the involved in skin wounding have specific properties, some principle of protein-dye binding. Analyt. Biochcm. 72, 248—254. of which resemble smooth muscle cells (Skalli and BULLOUQH, W. S. (1970). Epithelial Repair and Regeneration (eds. J. E. Gabbiani, 1988). Their origin in wounds appears to be local Dunphy and W. Van Winkle), pp. 35-46. New York- McGraw Hill. CLOWES, A. W., CLOWES, M. M., KOCHER, O., ROPRAZ, P., CHAPONNIER, (Ross et al. 1970), and they are thought to be derived from C. AND GABBIANI, G. (1988). Arterial smooth muscle cells in vivo. fibroblasts (Skalli and Gabbiani, 1988, for review). A relationship between actin isoform expression and mitosis and their recent study of open cutaneous wounds in the rat has modulation by heparin. J. Cell Biol. 107, 1937-1945. suggested that fibroblasts in granulation tissue transform COUCHMAN, J. R. (1986). Rat hair follicle dermal papillae have an extracellular matrix containing basement membrane components. gradually into myofibroblasts, and during a restricted J. invest. Derm. 87, 762-767. period express a smooth muscle marker (Darby et al. 1990). DARBY, I., SKALJJ, O. AND GABBIANI, G. (1990). ^Smooth muscle actin is In superficial cutaneous wounds, a principal source of transiently expressed by myofibroblasts during experimental wound replacement skin epidermis comes from outer root sheath healing. Lab. Invest 63(1), 21-29. epidermal cells of the hair follicle (Eisen et al. 1955; DESMOULIERE, A., RUBBIA-BRANDT, L. AND GABBIANI, G. (1991). Modulation of actin isoform expression in cultured arterial smooth Bullough, 1970; Krawczyk, 1971; Pang et al. 1978). Given muscle cells by heparin and culture conditions. Arteriosclerosis (in the similarity between myofibroblast and dermal sheath press). cell characteristics, it is reasonable to suggest that the EISEN, A. Z., HOLYOKE, J. B. AND LOBITZ, W. C. (1955). Responses of the dermal sheath cell layer (previously shown to be a superficial portion of the human pilosebaceous apparatus to controlled injury. J. invest. Derm 25, 145-156. reservoir of papilla cells for the follicle, and situated GABBIANI, G., KOCHER, O., BLOOM, W. S., VANDERKERKHOVE, J. AND adjacent to the outer root sheath epidermal cells) might be WEBER, K. (1984). Actin expression in smooth muscle cells of rat a specific source of myofibroblasts in cutaneous wounds. aortic intimal thickening, human atheromatous plaque and cultured This hypothesis is currently the subject of study. rat aortic media. J. elm. Invest. 73, 148-152 HORNE, K. A., JAHODA, C. A. B. AND OLIVER, R. F. (1986). Whisker growth induced by implantation of cultured vibrissa dermal papilla Concerning hair follicles, it is worth considering that cells in the adult rat J. Embryol. exp. Morph. 97, 111-124. minoxidil, the only human hair growth-promoting com- JAHODA, C. A. B., HORNE, K. A., MAUCEE, A., BARD, S. AND SENQEL, P. pound to have had reported success (Katz, 1988), was (1991). Cellular and extracellular involvement in lower rat vibrissa developed as an antihypertensive drug, with its principal follicle regeneration. Development (in press) mode of action on the vascular system (Zinns, 1988), and a JAHODA, C. A. B., HORNE, K. A. AND OLIVER, R. F. (1984). Induction of hair growth by implantation of cultured dermal papilla cells. Nature known capacity in vivo to relax vascular smooth muscle 311, 560-562. yia an active metabolite, minoxidil sulphate (Meisheri et JAHODA, C. A. B. AND OLIVER, R. F. (1981). The growth of vibrissa al. 1988). More intriguingly, smooth muscle cell features dermal papilla cells in vitro. Br J. Derm. 105, 623-627. have been described in the fibroblasts of normal organs JAHODA, C. A. B. AND OLIVER, R. F. (1984). Vibrissa dermal papilla cell aggregative behaviour in vivo and m vitro. J. Embryol. exp Morph. where some tissue reorganization occurs either continu- 79, 211-224. ously or periodically (Sappino et al. 1990, for a review), and JAHODA, C. A. B., OLIVER, R. F., FORRESTER, J. C. AND HORNE, K. A. where contractile function might be involved. One of us (1989). Regeneration in human hair follicles grafted onto athymic (Reynolds et al. unpublished data) has observed changes in mice. J. invest Derm. 93<3), 452. KATZ, H. I. (1988). Topical Minoxidil: Review of Efficacy. In Clinics in smooth muscle a^actin expression in the dermal sheath Dermatology. Androgenic Alopecia From Empiricism to Knowledge (ed. during the hair growth cycle, and has suggested that the R. L. de Villez), pp 195-199. Philadelphia: Lippincott.

Smooth muscle a-actin in hair follicle dermis 635 KLIOMAN, A L. (1988). The comparative histopathology of male-pattern scanning and transmision electron microscopic study. Am. J. Anat. baldness and senescent baldness. In Clinics m Dermatology. 153, 177-192. Androgenic Alopecia From Empiricism to Knowledge (ed. R. L. de REYNOLDS, A. J. (1989). In vivo and in vitro studies of isolated and Villez), pp. 108-118. Philadelphia: Lippincott. interacting dermal and epidermal components of the integument. KOCHKR, 0. AND GABBIANI, G. (1987). Analysis of (^smooth muscle actin Ph.D. thesis. University of Dundee. mRNA expression in rat aortic smooth-muscle cells using a specific Ross, R., EVERETT, N. B. AND TYLER, R. (1970). Wound healing and cDNA probe. Differentiation 34, 201-209. collagen formation. VI. The origin of the wound fibroblast studied in KOCHKE, 0., SKALLI, O., BLOOM, W S. AND GABBIANI, G. (1984). parabiosis. J. Cell Biol. 44, 645-654. Cytoskeleton of rat aortic smooth muscle cells. Normal conditions and SAPPINO, A. P., SCHURCH, W. AND GABBIANI, G. (1990). The experimental intimal thickening. Lab. Invest. SO, 645-652. differentiation repertoire of flbroblaatic cells: expression of KRAWCZYK, W. S (1971). A pattern of epidermal migration during cytoskeletal proteins as marker of phenotypic modulations. Lab. wound healing. J. Cell Bwl. 49, 247-263. Invest. 63, 144-161. MSISHERI, K. D , CIPKTJS, L. A AND TAYLOR, C. J. (1988). Mechanisms of SCHMITT-GRAFT, A., PAU, H., SPAHR, R., PIPER, H , SKALLI, O. AND action of minoxidil sulfate-induced vasodilation: a role for increased GABBIANI, G. (1990). Appearance of alpha-smooth muscle actin in K+ permeability. J. Pharm. exp. Ther. 245, 751-760. human eye lens cells of anterior capsular cataract and in cultured MESSENGER, A. G (1984). The culture of dermal papilla cells from bovine lens forming cells. Differentiation 43, 115-122. human hair follicles. Br. J. Derm. 110, 685-689. SKNGKL, P. (1986). Epidermal—dermal interaction. In Biology of the MESSENGER, A. G. (1989). Isolation, culture and in vitro behaviour of Integument, vol. 2, Vertebrates (ed. J Bereiter-Hahn, A. G. Matoltsy, cells isolated from papillae of human hair follicles. In Trends in and K. S. Richards), pp. 374-408. Berlin, Heidelberg: Springer-Verlag. Human Hair Growth and Alopecia Research (ed. D. Van Neste, J. M. SKALLI, O. AND GABBIANI, G. (1988). The biology of the myofibroblast Lachapelle and J. L. Antoine), pp. 57-66. London- Kluwer. relationship to wound contraction and fibrocontractive disease. In The MESSENGER, A. G., SENIOR, H. J. AND BLEEHEN, S. S. (1986). The in vitro Molecular and Cellular Biology of Wound Repair (ed. R A. F. Clark properties of dermal papilla cell lines established from human hair and P. M. Henson), pp.373-402. New York, London: Plenum press. follicles. Br. J. Derm. 114, 426-430. SKALLI, 0., ROPRAZ, P., TRZECIAK, A., BKNZONANA, G., GILLESSEN, D OTARRELL, P. H. (1975). High resolution two-dimensional AND GABBIANI, G. (1986). A monoclonal antibody against cr-Bmooth electrophoresis of proteins. J. biol. Chem. 250, 4007-4021. muscle actin: A new probe for smooth muscle differentiation. J. Cell OLIVER, R. F. (1966a). Whisker growth after removal of dermal papilla Biol. 103 (no. 6, Pt 2), 2787-2796. and lengths of the follicle in the hooded rat. J. Embryol. exp. Morph. SKALLI, 0., VANDEKERCKOVE, J. AND GABBIANI, G. (1987). Actin-isoform 15, 331-347. pattern as a marker of normal or pathological smooth muscle and OLIVER, R. F. (19666). Histological studies of whisker regeneration in fibroblastic tissues. Differentiation 33, 232-238. the hooded rat J. Embryol. exp. Morph. 18, 43-51. WESSELB, N. K. AND ROESSNER, K. D. (1965). Non-proliferation in dermal OLIVER, R. F. (1967). The experimental induction of whisker growth in condensations of mouse vibrissae and pelage . Devi Biol. 12, the hooded rat by implantation of dermal papillae. J. Embryol. exp. 419-433. Morph. 18, 43-51 ZINNS, G. R. (1988). The history of the development of minoxidil. In OLIVER, R F. (1970). The induction of follicle formation in the adult Clinics in Dermatology. Androgenic Alopecia From Empiricism to hooded rat by vibrissa dermal papillae. J. Embryol. exp. Morph. 23, Knowledge (ed. R. L. de Villez), pp. 132-143. Philadelphia: Lippincott. 219-236. PANG, S. C, DANIELS, W. H. AND BUCK, R. C. (1978). Epidermal migration during the healing of suction blisters in rat skin: a (.Received 13 December 1991 - Accepted, in revised form, 5 April 1991)

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