The secret life of the hair follicle.

The mammalian hair follicle is a treasure waiting to be discovered by more molecular geneticists. How can a tiny cluster of apparently uniform epithelial cells, adjacent to a tiny cluster of uniform mesenchymal cells, give rise to five or six concentric cylinders, each of which is composed of cells of a distinctive type that synthesize their own distinctive set of proteins? There is now evidence that several growth factors, cell adhesion molecules and other molecules play important roles in the regulation of this minute organ.

The stability of vitamin A-induced metaplasia of mouse vibrissa follicles in vitro.

Tests were made of the stability of the previously described glandular morphogenesis and mucous metaplasia of embryonic mouse vibrissa follicles produced in vitro by excess vitamin A. The changes in individual follicles were observed in living tissue explants and serial sections. Upper lip skin of 13.5-day embryos underwent budding from vibrissa follicles to form branching glands which secreted mucus after 10--14 days in medium containing 4.7 microgram/ml retinol. If this medium was replaced with standard medium after 7 days, glandular morphogenesis and metaplasia continued unaffected, although some hair follicle bases returned to their original morphogenetic program. Similar results were obtained with skin of 13-day embryos treated with vitamin A (6.0 MG/ML) for only 3 days. The moderate degree of glandular morphogenesis in 15-day skin with vitamin A (4.7 microgram/ml) was not altered by the simultaneous addition of a high dose of cortisol (18 microgram/ml). It is suggested that the initiation of glandular morphogenesis of follicles differs from the initiation of mucous metaplasia in embryonic chick epidermis by vitamin A, in that it resembles a new secondary embryonic induction rather than a modulation of the epithelium.

Basal lamina changes during tissue interactions in hair follicles--an in vitro study of normal dermal papillae and vitamin A-induced glandular morphogenesis.

Skin pieces from 14-day fetal mice were cultivated for 1-10 days prior to fixation and sectioning. Subsequently, sections were studied by light and transmission electron microscopy. In a standard medium the lateral hair follicle walls showed progressive maturation of the basal lamina, while the hair matrix, at the time of a known tissue interaction, showed the formation of gaps in the basal lamina, with heterotypic cell contacts through the gaps. In a vitamin-A enriched medium similar changes occurred, not only at the hair matrix, but also at lateral follicle walls, at the sites of, and prior to, budding and glandular morphogenesis. This study shows that the induction of hair matrix by dermal papilla may perhaps be added to the list of normal tissue interactions in which heterotypic cell contacts occur. It also suggests that vitamin-A induced glandular morphogenesis might come about through a mechanism resembling a normal tissue interaction.

Effects of steroid hormones on developing mouse skin in vitro.

Pieces of skin from 13-5- to 15-day-old foetal mice were grown in organ culture in a biological medium with or without the addition of hormonal steroidsmcortisol (7-5 mug/ml) caused thinning of the non-cornified epidermis and flattening of the stratum granulosum after 3 days. By 6 days the epidermis was thinner and hair follicles were regressing, and these changes continued up to 12 days. Administration of corticosterone (5 mug/ml) also produced thinning of the epidermis and regression of the follicles after 6 days. Good differentiation of epidermis and hair follicles was obtained when testosterone (100 mug/ml) was added to the medium. The non-cornified epidermal layers were similar to those of control cultures at 3 days but less than half as thick at 6 days. Hair follicles differentiated as rapidly in medium containing testosterone as in normal medium, but, unlike in the latter medium, also developed sebaceous gland anlagen at 6 days. Some explants in testosterone medium showed signs of sebaceous cell differentiation at 9 days.

A new type of lesion associated with severe fur damage in Canadian ranch foxes and an investigation of possible causes.

In the silver fox, as in its wild ancestor, the red fox (Vulpes vulpes L.), the annual growing phase (anagen) of guard hair follicles occupies at least four months. Severe damage to the hair coat near the end of this growing period was reported in 1985 on many ranches in New Brunswick and Nova Scotia. A histological analysis of serial sections of skin biopsies showed a marked increase in nuclear aberrations in the hair matrix of anagen guard hair follicles. These nuclear aberrations indicated that cells were undergoing apoptosis, a controlled form of cell death. Tissues from affected and unaffected foxes for histological and toxicological analysis, as well as other data, were obtained during visits to 26 ranches in 1986 and 34 ranches in 1987. Histological sections of the 1987 skin samples showed the mean percentage of nuclear aberrations in 43 unaffected foxes to be 0.08 +/- 0.01 (SEM), while that for 49 affected foxes was 0.51 +/- 0.23. The four foxes with the most severe coat damage also had the highest incidences of guard hair matrix cells with nuclear aberrations, ranging from 20 to 100 times greater than the mean for unaffected foxes. The mitotic index of the hair matrix, which normally remains fairly constant during the hair growth phase, was similar for unaffected and affected foxes (1.83 +/- 0.06 and 1.97 +/- 0.07 respectively). Although our analyses of field data have not established a specific environmental factor associated with increased nuclear aberrations, the possible involvement of toxic agents in follicle damage may warrant further investigation.

Normal levels of vitamin A in tissues of the Syrian hamster (Mesocricetus auratus) determined colorimetrically.

Vitamin A levels in tissues of 20 normal adult hamsters on a standard diet were measured colorimetrically. No significant difference between male and female animals was found for any of the tissues sampled. The mean vitamin A value for blood plasma in 20 animals was 53.4 micrograms/dl. Mean values for liver, kidneys, flank skin and cheek pouch were 813, 1.29, 1.84 and 1.31 mg/g wet weight, respectively. The vitamin assay was less suitable for small organs such as trachea.

In vitro development of secondary blastodiscs from dispersed blastoderm cells of Gallus domesticus.

A study of the in vitro growth of embryonic structures from dispersed blastoderm cells is reported. The specific type of blastoderm cells with the capability of growing on the coverslip developed into the discoidal embryonic structures resembling those of avian species. The glass surface was apparently an initiator of differentiation into at least three types of cells specifically distributed in the blastodisc. Groups of structures formed were evaluated at 6, 12, 24 and 36 hours to study the developmental pattern in vitro and to estimate the number of cells per structure. Microscopic examination of the area pellucida revealed that all three basic germ layers were established after 24 hours of incubation in vitro. The 36 hour stage was represented by bulky growth of mesodermal-like cells and changes in hypoblast layer where some of the cells degenerated and some were transformed to mesenchymal spindle-like cells.

The use of retinoids as probes for analyzing morphogenesis of glands from epithelial tissues.

Thirty-five years ago Honor Fell and Edward Mellanby were studying effects of high doses of vitamin A on skeletal development in chick embryos when they noticed that a piece of epidermis, accidentally included in an organ culture, had undergone mucous metaplasia. Further studies by Fell and others eventually led to an understanding of the important role of vitamin A in modulating epithelia in vivo. Fifteen years later another organ culture experiment showed me that excess vitamin A could also initiate the morphogenesis of branching and mucus-secreting glands from developing vibrissa follicles in upper lip skin of embryonic mice. Since then our group has shown that induction of this novel structure by naturally occurring retinoids resembles a normal embryonic induction in that it is stage-dependent, time-dependent, and irreversible. Tissue separation and recombination studies showed that isolated upper lip epidermis can form these glands when combined with retinoid-treated upper lip dermis. Untreated mouse epidermis can form similar glands after combination with chick dermis containing higher retinoid levels. The hamster cheek pouch, normally devoid of glandular structures, can also form mucous glands when treated with a retinoid, either in vivo or in vitro. Recombination studies in organ culture have now shown that mesenchyme exposed to retinoid is essential for gland morphogenesis from pouch epithelium. Evidence is accumulating that retinoic acid may even be the active morphogen in some normally developing systems.

Morphological changes at the basement membrane during some tissue interactions in the integument.

Changes at the basement membrane have been reported at the time of tissue interactions during the development of a number of integumentary organs. During the development of vibrissa follicles of the mouse in vitro, gaps appeared in the lamina densa surrounding the dermal papilla at the time of the second, specific dermal message for follicle differentiation. Direct contacts between epithelial and mesenchymal cells were made through these gaps. This area has now been studied in vivo over the same period of follicle development. In addition, the epithelial part of the follicle has been separated from the dermal papilla by (i) enzyme treatment which destroys the lamina densa and (ii) chelating agents which leave the lamina densa attached to the dermal papilla. The resulting surfaces of epithelium and dermal papilla were examined by scanning and transmission electron microscopy. Small gaps first appeared in the lamina densa at the same substage of follicle development as in vitro and were present at the next substage. Processes from the dermal papilla mesenchyme cells traversed these gaps and made close contacts with the epithelial cells just prior to their differentiation into inner root sheath and hair. Instructive interaction through cell surfaces is suggested.

Effects of intradermally injected and topically applied mouse epidermal growth factor on wool growth, skin and wool follicles of merino sheep.

Twice daily intradermal (ID) injections of mouse epidermal growth factor (mEGF) in sterile saline for 1-4 days into delineated areas of skin of Merino sheep produced dose-dependent changes in wool follicles and fibres, ranging from slight reduction in follicle bulb size and transient disturbance of cuticle formation on some fibres to the induction of catagen of follicles and shedding of fibres with distorted, tapered ends. Regeneration of follicles commenced by day 7. By contrast, ID injections of saline did not affect follicle activity. The epidermis became thicker and more parakeratotic after multiple injections of mEGF than after injection of saline, but was almost normal again by day 14. Persistent small increases in sebaceous gland size, additional to those induced by ID injections of saline, and delayed small increases in sweat gland size also occurred after multiple injections of mEGF. Daily topical applications of mEGF in 50% (v/v) aqueous propylene glycol 5 days each week for 4 weeks did not affect wool growth or the follicles and other skin components. The only effect observed, due to application of the aqueous propylene glycol, was an increase in the number of layers of cornified cells in the stratum corneum of the epidermis, with the cells arranged in clearly discernible stacks. The effects produced by ID injections of mEGF indicate that mEGF acts directly on the pilosebaceous and epidermal components of skin.

Changing patterns of cell adhesion molecules during mouse pelage hair follicle development. 1. Follicle morphogenesis in wild-type mice.

The morphogenesis of hairs is initiated and maintained by reciprocal interactions between groups of epithelial and mesenchymal cells. To examine whether cell adhesion molecules play a role in this process, prenatal distribution patterns of various cell adhesion molecules were studied during hair follicle morphogenesis in the dorsal skin of C57BL mouse embryos, using monoclonal antibodies. E-cadherin was present on all epithelial cells of the skin when the ectoderm gave rise to periderm and epidermis. E-cadherin was reduced in the follicle placodes and hair plugs, then disappeared from the presumptive hair matrix of elongating follicles. P-cadherin was initially present on all cells of periderm and epidermis and was later retained at a reduced level in the basal epidermal layer. P-cadherin was prominent in all follicle placodes and hair plugs and in the presumptive hair matrix of elongating follicles. N-CAM was present on all mesenchymal cells of the presumptive dermis at the prefollicle stage, then temporarily restricted to a few cells just below the dermal-epithelial junction. Later, N-CAM reappeared in the interfollicular mesenchyme and was prominent in the mesenchymal sheath and dermal papilla of elongating follicles. In addition, N-CAM was expressed in the hair plugs, then became progressively restricted to the upper caudal part of the elongating follicles. The results suggest that the main role of cell adhesion molecules is to mould the follicle by relaxing or reinforcing cell contacts in areas of increased morphogenetic activity.

Changing patterns of cell adhesion molecules during mouse pelage hair follicle development. 2. Follicle morphogenesis in the hair mutants, Tabby and downy.

Wild-type mice have three main types of hair in their pelage: tylotrichs, awls and zigzags. Tabby mice have a yellowish coat consisting of awls only, whereas downy mice have a sparse grayish coat consisting of unusually fine hairs. The spatial and temporal distribution of cell adhesion molecules (CAMs) during hair follicle morphogenesis was investigated in the mutants and compared with that in nonmutant mice. In Tabby embryos, awl follicles developed normally and showed normal immunostaining patterns for E-cadherin, P-cadherin and N-CAM. Prior to follicle initiation, however, some deviations from normal skin morphology and staining patterns indicated a delay in the development of the basal epidermal layer. On the other hand, the stratum corneum was formed prematurely. Therefore, the lack of tylotrich and zigzag follicles in Tabby mice might be explained by a general defect in epidermal development rather than by abnormal CAM expression. In downy embryos, tylotrich and awl follicles were initiated within the normal time periods, but elongation and differentiation of most follicles were abnormal. At birth, most follicles were small and/or severely deformed but showed normal CAM expression patterns. Extreme distortion and disorientation of follicles seemed to be associated with disintegration of the dermal papilla and abnormal mesenchymal cell condensations between the follicles. This suggests that abnormal hair development in downy mice might result from a defect in dermal rather than epidermal components of the skin.

Excess retinoid acts through the stroma to produce mucous glands from newborn hamster cheek pouch in vitro.

Many morphogenetic processes are modified or initiated by retinoids. The cheek pouch of the newborn hamster can be induced to develop mucous glands in vitro by adding excess retinoid. The objective of this study was to determine whether the retinoid acted through the epithelium or the mesenchymal stroma. Explants of cheek pouch were grown for 7 days in either standard medium, or medium supplemented with 6 micrograms/ml retinyl acetate (RAc; 1.8 x 10(-5) M). After separation of most explants into epithelium and mesenchyme by trypsinization, the separated tissues were recombined in all possible ways and cultured for a further 1-2 weeks in standard medium. All explants were analysed histologically and/or histochemically from complete serial paraffin sections. No glands were formed in 30 recombinants containing stroma that had not been exposed to RAc, but four of 25 recombinants containing previously exposed stroma had glands, as well as four of 18 unseparated explants exposed to RAc. Exposure of epithelium to RAc did not result in the incidence of glands. It was concluded that RAc acting through the stroma was responsible for the instructive interaction with the epithelium for gland formation. A molecular mechanism is suggested.

Stability of the glandular morphogenesis produced by retinoids in the newborn hamster cheek pouch in vitro.

Retinoids can induce alterations in differentiation and morphogenesis in the hamster cheek pouch. In order to determine the stability of these changes, explants of neonatal pouch were exposed to 6 micrograms/ml of either retinyl acetate (RAc: 1.8 x 10(-5) M) or all-trans retinoic acid (RA: 2.0 x 10(-5) M) for an initial 3 of 7 days, out of a total of 21 days in organ culture. Three days of RAc or RA caused a delay in the differentiation and keratinization of the epithelium at least up to day 7 of culture. Additionally, two out of ten explants exposed to RA showed small downgrowths of epithelium into the stroma at 7 or 14 days. Seven days of exposure to either retinoid led to inhibition of epithelial keratinization, and produced a mucous metaplasia which was still seen at the end of the 21-day culture period. Periodic acid-Schiff (PAS)-positive, diastase-resistant material was present in the metaplastic epithelium, in intercellular, and in some instances, intracellular locations. An excess of either RAc or RA, for 7 days, induced persistent glandlike downgrowths of epithelium, suggesting that a stable alteration in the developmental program of the epithelium may have occurred. Many of these downgrowths possessed a lumen which was lined by cuboidal epithelium and contained PAS-positive, diastase-resistant secretory material. RA appeared more potent than RAc in inhibiting keratinization, in producing a mucous metaplasia, and in initiating glandlike downgrowths. The persistence of glandular downgrowths suggests that retinoids, either directly or indirectly, act in a manner similar to that of an embryonic inductor.

Histochemical evidence of mucosubstances in the metaplastic epidermis and hair follicles produced in vitro in the presence of excess vitamin A.

Pieces of upper lip and lower jaw from 13-day old mouse embryos grown in organ culture with excess vitamin A showed metaplastic changes in epidermis and hair follicles after 7 to 21 days. Changes were less marked in upper lip explants from 15-day old embryos. Areas of keratinizing epidermis showed a much higher incidence of Alcian blue-positive bodies when excess vitamin A was present. Histochemical tests with Alcian blue at critical electrolyte concentrations indicated moderately sulfated acidic mucosubstances in these bodies. Other patches of epidermis were transformed into stratified cuboidal epithelium producing PAS-positive, Alcian blue-negative, mucosubstances. The hair follicles, transformed by vitamin A into glands, developed three types of epithelium with apical granules, luminal borders and occasional goblet cells, all showing mucosubstances. Tests suggest that both metaplastic epidermis and glands secrete either neutral mucosubstances or non-sulfated acidic mucosubstances. Another response to vitamin A was a rapid loss of glycogen, particularly in hair follicles and the epidermal basal layer. It was concluded that excess vitamin A alters not only the morphogenesis but also the distribution and synthesis of polysaccharides in developing skin.

Ciliated and secretory epidermis produced from embryonic mammalian skin in organ culture by vitamin A.

When skin from the upper lip of 12-day embryonic mice was grown for ten days in organ culture with 5.7 mug retinol added per ml of biological medium, keratinization was suppressed and a ciliated and secretory epithelium was produced. Ultrastructural features of this epithelium are described. At this very early stage mouse epidermis is thus similar of chick epidermis in its ability to undergo radical metaplasia in response to vitamin A.