CELL KINETICS IN THE SEBACEOUS GLANDS OF THE MOUSE. I. THE GLANDS IN RESTING SKIN

Cell proliferation, differentiation and migration have been studied in the sebaceous glands of DBA-2 mice in the resting (telogen) phase of hair growth. Cells labelled by a single injection of tritiated thymidine start to leave the glands of adult male mice 5 days later. About 80% of the proliferative cells in the basal layer have a cell cycle time of 40 hr or less. In 18% of the proliferative cells G₁ is at least 4 days long and 16% have a G₂ phase longer than 17 hr. The S phase is about 7.5 hr long and cells spend at least 21 hr in the basal layer before migrating into the differentiating cell region. The glands of mature female and immature mice are smaller than those of the mature male. They have fewer, smaller cells and a much lower labelling index.     

HISTOLOGY AND CYTOCHEMISTRY OF HUMAN SKIN : IX. THE DISTRIBUTION OF NON-SPECIFIC ESTERASES

1. In the epidermis non-specific esterase activity outlines a strongly reactive band between the stratum granulosum and the stratum corneum. In the epidermis of the palm, there is no such esterase-rich band. 2. The outer sheath of active hair follicles has strong enzyme activity. The degenerating hair bulb in catagen follicles is very strongly reactive, and clusters of cells around the hair club in quiescent follicles are rich in enzyme activity. 3. Strong enzyme activity is found in young sebaceous cells, while decaying sebaceous cells and newly formed sebum are unreactive. Old sebum, however, is very intensely reactive. 4. Only the "dark" cells of eccrine sweat glands show a reaction; the "clear" cells are negative. 5. The cells of axillary apocrine glands abound in enzyme.     

Histology and cytochemistry of human skin. IX. The distribution of non-specific esterases

1. In the epidermis non-specific esterase activity outlines a strongly reactive band between the stratum granulosum and the stratum corneum. In the epidermis of the palm, there is no such esterase-rich band. 2. The outer sheath of active hair follicles has strong enzyme activity. The degenerating hair bulb in catagen follicles is very strongly reactive, and clusters of cells around the hair club in quiescent follicles are rich in enzyme activity. 3. Strong enzyme activity is found in young sebaceous cells, while decaying sebaceous cells and newly formed sebum are unreactive. Old sebum, however, is very intensely reactive. 4. Only the "dark" cells of eccrine sweat glands show a reaction; the "clear" cells are negative. 5. The cells of axillary apocrine glands abound in enzyme.     

Ornithine decarboxylase activity in relation to DNA synthesis in mouse interfollicular epidermis and hair follicles.

The relationship between ornithine decarboxylase (L-ornithine carboxylyase, EC 4.1.1.17) activity and DNA synthetic activity was studied in mouse epidermis. Interfollicular epidermis and hair follicles were investigated separately. It was found that, in hair follicles, the variations of DNA replicative activity, which are reflected in the cyclic growth of hair, are paralleled by corresponding changes in ornithine decarboxylase activity. In both interfollicular epidermis and hair follicles, stimulation of DNA synthetic activity by plucking of hair induced a rapid and marked increase in ornithine decarboxylase activity. The relationship of steady-state and induced ornithine decarboxylase activity to DNA synthetic activity was compared in hair follicles and interfollicular epidermis. A correlation between the activity of this enzyme and DNA replication was found thereby in each of these tissues.     

Manipulation of stem cell proliferation and lineage commitment: visualisation of label-retaining cells in wholemounts of mouse epidermis

Mammalian epidermis is maintained by stem cells that have the ability to self-renew and generate daughter cells that differentiate along the lineages of the hair follicles, interfollicular epidermis and sebaceous gland. As stem cells divide infrequently in adult mouse epidermis, they can be visualised as DNA label-retaining cells (LRC). With whole-mount labelling, we can examine large areas of interfollicular epidermis and many hair follicles simultaneously, enabling us to evaluate stem cell markers and examine the effects of different stimuli on the LRC population. LRC are not confined to the hair follicle, but also lie in sebaceous glands and interfollicular epidermis. LRC reside throughout the permanent region of the hair follicle, where they express keratin 15 and lie in a region of high alpha6beta4 integrin expression. LRC are not significantly depleted by successive hair growth cycles. They can, nevertheless, be stimulated to divide by treatment with phorbol ester, resulting in near complete loss of LRC within 12 days. Activation of Myc stimulates epidermal proliferation without depleting LRC and induces differentiation of sebocytes within the interfollicular epidermis. Expression of N-terminally truncated Lef1 to block beta-catenin signalling induces transdifferentiation of hair follicles into interfollicular epidermis and sebocytes and causes loss of LRC primarily through proliferation. We conclude that LRC are more sensitive to some proliferative stimuli than others and that changes in lineage can occur with or without recruitment of LRC into cycle.     

CELL PROLIFERATION KINETICS OF EPIDERMIS AND SEBACEOUS GLANDS IN RELATION TO CHALONE ACTION

Median S-phase lengths of pinna epidermis and sebaceous glands, and of epithelia from the oesophagus and under surface of the tongue of Albino Swiss S mice were estimated by the percentage labelled mitoses method (PLM). The 18.4 and 18.8 hr for the median length of S-phase for pinna epidermis and sebaceous glands respectively made it possible for these two tissues to be used experimentally for testing tissue specificity in chalone assay experiments. The 10.0 and 11.5 hr for oesophagus and tongue epithelium respectively made experimental design for chalone assay difficult when pinna epidermis was the target tissue. The results of the Labelling Index measured each hour throughout a 24-hr period showed no distinct single peaked diurnal rhythm for pinna epidermis and sebaceous glands. Instead a circadian rhythm with several small peaks occurred which would be expected if an S-phase of approximately 18 hr was imposed on the diurnal rhythm. This indicates that there may be very little change in the rate of DNA synthesis. The results are given for the assay in vivo of purified epidermal G₁ and G₂ chalones, and the 72–81% ethanol precipitate of pig skin from which they could be isolated. These experiments were performed over a time period which took into account the diurnal rhythm of activity of the mice as well as the S-phase lengths. Extrapolating the results with time of action of the chalone shows that the G₁ chalone acts at the point of entry into DNA synthesis and that the S-phase length was approximately 17 hr for both the pinna epidermis and sebaceous glands. This may be a more correct value since the PLM method overestimates the median S-phase length as it is known that in pinna skin the [³H]TdR is available to the tissues for 2 hr and true flash labelling does not take place. The previous reports that epidermal G₁ chalone acts some hours prior to entry into S-phase resulted from experiments on back skin where the S-phase is shorter and there is a pronounceddiurnal rhythm which could mask the chalone effect. The epidermal G, chalone had no effect on DNA synthesis even at different times in the circadian rhythm. Thus the circadian rhythms and S-phase lengths of the test tissues need to be considered when experiments are performed with chalones. Ideally, the target tissues selected for cell line specificity tests should have the same cell kinetics for the easier and more accurate assessment and interpretation of results. When the tissues have markedly different cell kinetics, experimental procedures and results need to be evaluated accordingly. The point of action of G, chalone can only be assessed if the effect is measured over the peak of incorporation of 13H]TdR into DNA. The results of the effects of skin extracts are analysed in relation to changes in the availability of i3H]TdR for the incorporation into DNA and to the possibility of there being two distinct populations of proliferating cells.     

Transient activation of beta-catenin signalling in adult mouse epidermis is sufficient to induce new hair follicles but continuous activation is required to maintain hair follicle tumours

When beta-catenin signalling is disturbed from mid-gestation onwards lineage commitment is profoundly altered in postnatal mouse epidermis. We have investigated whether adult epidermis has the capacity for beta-catenin-induced lineage conversion without prior embryonic priming. We fused N-terminally truncated, stabilised beta-catenin to the ligand-binding domain of a mutant oestrogen receptor (DeltaNbeta-cateninER). DeltaNbeta-cateninER was expressed in the epidermis of transgenic mice under the control of the keratin 14 promoter and beta-catenin activity was induced in adult epidermis by topical application of 4-hydroxytamoxifen (4OHT). Within 7 days of daily 4OHT treatment resting hair follicles were recruited into the hair growth cycle and epithelial outgrowths formed from existing hair follicles and from interfollicular epidermis. The outgrowths expressed Sonic hedgehog, Patched and markers of hair follicle differentiation, indicative of de novo follicle formation. The interfollicular epidermal differentiation program was largely unaffected but after an initial wave of sebaceous gland duplication sebocyte differentiation was inhibited. A single application of 4OHT was as effective as repeated doses in inducing new follicles and growth of existing follicles. Treatment of epidermis with 4OHT for 21 days resulted in conversion of hair follicles to benign tumours resembling trichofolliculomas. The tumours were dependent on continuous activation of beta-catenin and by 28 days after removal of the drug they had largely regressed. We conclude that interfollicular epidermis and sebaceous glands retain the ability to be reprogrammed in adult life and that continuous beta-catenin signalling is required to maintain hair follicle tumours.     

Localization of sex steroid receptors in human skin

Sex steroid hormones are involved in regulation of skin development and functions as well as in some skin pathological events. To determine the sites of action of estrogens, androgens and progestins, studies have been performed during the recent years to accurately localize receptors for each steroid hormone in human skin. Androgen receptors (AR) have been localized in most keratinocytes in epidermis. In the dermis, AR was detected in about 10% of fibroblasts. In sebaceous glands, AR was observed in both basal cells and sebocytes. In hair follicles, AR expression was restricted to dermal papillar cells. In eccrine sweat glands, only few secretory cells were observed to express AR. Estrogen receptor (ER) alpha was poorly expressing, being restricted to sebocytes. In contrast, ERbeta was found to be highly expressed in the epidermis, sebaceous glands (basal cells and sebocytes) and eccrine sweat glands. In the hair follicle, ERbeta is widely expressed with strong nuclear staining in dermal papilla cells, inner sheath cells, matrix cells and outer sheath cells including the buldge region. Progesterone receptors (PR) staining was found in nuclei of some keratinocytes and in nuclei of basal cells and sebocytes in sebaceous glands. PR nuclear staining was also observed in dermal papilla cells of hair follicles and in eccrine sweat glands. This information on the differential localization of sex steroid receptors in human skin should be of great help for future investigation on the specific role of each steroid on skin and its appendages.     

A histological study on the skin and hairs of PC (poor coat) mice

Light microscopic examinations were done on the skin and hairs of PC (poor coat) mice, maintained as an inbred strain at the National Institute of Health, Japan. The structures of the epidermis, dermis, hair root sheath and the sebaceous glands were normal. Hair bulbs and hair papillae were poorly developed at anagen stage of hair cycle. Having scanty medulla, the hairs were thin and short. The hair cuticle appeared normal. These findings suggest that the defective hair growth in PC mice is caused by deficiencies in cell differentiation and/or proliferation in the hair matrix.     

Comparative responsiveness of supracaudal and other sebaceous glands in male and female guinea pigs to hormones

Different types of sebaceous glands in guinea pigs were chosen to study their comparative responsiveness to steroid hormones. Glands selected were (1) Sebaceous glands associated with rudimentary hair in the supracaudal gland; (2) Free sebaceous glands of the nipple; (3) Sebaceous glands associated with the hair. The results showed distinct differences in sensitivity among these different sebaceous glands and according to the sex of the experimental animal. The most responsive of the glands in males was the supracaudal gland; in the female the most responsive glands were the sebaceous glands of the nipple. Sebaceous glands associated with normal hair were relatively insensitive to changes in hormonal level. In all cases testosterone propionate was more potent in stimulating any of the sebaceous glands than progesterone. After gonadectomy, various sebaceous glands showed unequal states of depletion due to the different rates of differentiation and disintegration of their cells into sebum.     

Normal fur development and sebum production depends on fatty acid 2-hydroxylase expression in sebaceous glands

2-Hydroxylated fatty acid (HFA)-containing sphingolipids are abundant in mammalian skin and are believed to play a role in the formation of the epidermal barrier. Fatty acid 2-hydroxylase (FA2H), required for the synthesis of 2-hydroxylated sphingolipids in various organs, is highly expressed in skin, and previous in vitro studies demonstrated its role in the synthesis of HFA sphingolipids in human keratinocytes. Unexpectedly, however, mice deficient in FA2H did not show significant changes in their epidermal HFA sphingolipids. Expression of FA2H in murine skin was restricted to the sebaceous glands, where it was required for synthesis of 2-hydroxylated glucosylceramide and a fraction of type II wax diesters. Absence of FA2H resulted in hyperproliferation of sebocytes and enlarged sebaceous glands during hair follicle morphogenesis and anagen (active growth phase) in adult mice. This was accompanied by a significant up-regulation of the epidermal growth factor receptor ligand epigen in sebocytes. Loss of FA2H significantly altered the composition and physicochemical properties of sebum, which often blocked the hair canal, apparently causing a delay in the hair fiber exit. Furthermore, mice lacking FA2H displayed a cycling alopecia with hair loss in telogen. These results underline the importance of the sebaceous glands and suggest a role of specific sebaceous gland or sebum lipids, synthesized by FA2H, in the hair follicle homeostasis.     

Phospholipase Cdelta1 is required for skin stem cell lineage commitment

Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in phosphoinositide turnover and is involved in a variety of physiological functions. Here we report that PLCdelta(1)-deficient mice undergo progressive hair loss in the first postnatal hair cycle. Epidermal hyperplasia was observed, and many hairs in the skin of PLCdelta(1)-deficient mice failed to penetrate the epidermis and became zigzagged owing to occlusion of the hair canal. Two major downstream signals of PLC, calcium elevation and protein kinase C activation, were impaired in the keratinocytes and skin of PLCdelta(1)-deficient mice. In addition, many cysts that had remarkable similarities to interfollicular epidermis, as well as hyperplasia of sebaceous glands, were observed. Furthermore, PLCdelta(1)-deficient mice developed spontaneous skin tumors that had characteristics of both interfollicular epidermis and sebaceous glands. From these results, we conclude that PLCdelta(1) is required for skin stem cell lineage commitment.     

Demonstration of β-glucan receptors in the skin of aquatic mammals—a preliminary report

Using immunohistochemistry, the study clearly demonstrates three important β-glucan receptors (Ficolin/P35, MBL, Dectin-1; members of the lectin-complement pathway of innate immunity) in the integument of six marine and freshwater aquatic mammals (Northern fur seal, Common seal, Walrus, Coypu, Capybara, Otter), but only weakly in two dolphin species. Most of the non-dolphin mammals exhibited strong reactions, especially with regard to the skin glands (tubular apocrine glands, sebaceous glands), for L-Ficolin/P35 and MBL. Distinct reaction staining could also be observed in the epidermis and the outer epithelial sheath of primary hair follicles. Positive Dectin-1 staining was limited to secretory cells of the apocrine tubular glands, and to peripheral and central cells of sebaceous glands of the seals. The Capybara was the only animal to show a clear Dectin reaction in the epidermis (stratum granulosum). The findings are discussed with regard to the constant and high microbial challenge of the skin in the aquatic medium, and variations in hair density of the animals.     

Estradiol distribution and penetration in rat skin after topical application, studied by high resolution autoradiography

Transdermal pathways and targets in the skin for estradiol were investigated using dry-mount autoradiography. 3H-estradiol-17 beta was applied at doses of 30.1 pmol, 120.4 pmol and 301 pmol/cm2 to shaved rat skin in the dorsal neck region. Vehicles were DMSO, ethylene glycol or sesame oil. After 2 h of topical treatment with 30.1 pmol 3H-estradiol x cm-2 dissolved in DMSO a distinct cellular distribution was apparent. Target cells with concentrations of radioactivity were found in epidermis, sebaceous glands, dermal papillae of hair and fibroblasts. After treatment with 120.4 and 301 pmol/cm2, a penetration gradient of radioactivity was recognizable however it masked specific cellular and subcellular uptake. The stratum corneum accumulated and retained radioactivity, apparently forming a depot for the hormone. Strong concentration and retention of the hormone was conspicuous in sebaceous glands for more than 24 h, suggesting that sebaceous glands serve as a second storage site for the hormone. In all autoradiograms two penetration pathways to the dermis were visible: one through the stratum corneum and epidermis, the other through the hair canals and hair sheaths.     

Morphogenesis and renewal of hair follicles from adult multipotent stem cells

The upper region of the outer root sheath of vibrissal follicles of adult mice contains multipotent stem cells that respond to morphogenetic signals to generate multiple hair follicles, sebaceous glands, and epidermis, i.e., all the lineages of the hairy skin. At the time when hair production ceases and when the lower region of the follicle undergoes major structural changes, the lower region contains a significant number of clonogenic keratinocytes, and can then respond to morphogenetic signals. This demonstrates that multipotent stem cells migrate to the root of the follicle to produce whisker growth. Moreover, our results indicate that the clonogenic keratinocytes are closely related, if not identical, to the multipotent stem cells, and that the regulation of whisker growth necessitates a precise control of stem cell trafficking.     

Glutamate transporter Slc1a3 mediates inter‐niche stem cell activation during skin growth

Tissues contain distinct stem cell niches, but whether cell turnover is coordinated between niches during growth is unknown. Here, we report that in mouse skin, hair growth is accompanied by sebaceous gland and interfollicular epidermis expansion. During hair growth, cells in the bulge and outer root sheath temporarily upregulate the glutamate transporter SLC1A3, and the number of SLC1A3⁺ basal cells in interfollicular epidermis and sebaceous gland increases. Fate mapping of SLC1A3⁺ cells in mice revealed transient expression in proliferating stem/progenitor cells in all three niches. Deletion of slc1a3 delays hair follicle anagen entry, uncouples interfollicular epidermis and sebaceous gland expansion from the hair cycle, and leads to reduced fur density in aged mice, indicating a role of SLC1A3 in stem/progenitor cell activation. Modulation of metabotropic glutamate receptor 5 activity mimics the effects of SLC1A3 deletion or inhibition. These data reveal that stem/progenitor cell activation is synchronized over distinct niches during growth and identify SLC1A3 as a general marker and effector of activated epithelial stem/progenitor cells throughout the skin.     

Multiple classes of stem cells in cutaneous epithelium: a lineage analysis of adult mouse skin

Continuous renewal of the epidermis and its appendages throughout life depends on the proliferation of a distinct population of cells called stem cells. We have used in situ retrovirus-mediated gene transfer to genetically mark cutaneous epithelial stem cells of adolescent mice, and have followed the fate of the marked progeny after at least 37 epidermal turnovers and five cycles of depilation-induced hair growth. Histological examination of serial sections of labeled pilosebaceous units demonstrated a complex cell lineage. In most instances, labeled cells were confined to one or more follicular compartments or solely to sebaceous glands. Labeled keratinocytes in interfollicular epidermis were confined to distinct columnar units representing epidermal proliferative units. The contribution of hair follicles to the epidermis was limited to a small rim of epidermis at the margin of the follicle, indicating that long term maintenance of interfollicular epidermis was independent of follicle-derived cells. Our results indicate the presence of multiple stem cells in cutaneous epithelium, some with restricted lineages in the absence of major injury.     

The skin of primates. XXIX. The skin of the pigmy bushbaby (Galago demidovii)

The skin of the pigmy bushbaby (Galago demidovii), the smallest existing prosimian, is largely similar to that of the other African Lorisidae, although this animal has certain peculiar features. The very thin epidermis contains alkaline phosphatase-reactive dendritic cells which resemble those in the other bushbabies and the potto. The hair follicles of this animal are similar to those of the lesser bushbaby, while the sebaceous glands are different from those of the other bushbabies in being reactive for alkaline phosphatase. The histological and histochemical properties of the sweat glands are similar to those of the other bushbabies.     

Mechanisms regulating epidermal stem cells

The skin epidermis contains different appendages such as the hair follicle and the sebaceous glands. Recent studies demonstrated that several types of stem cells (SCs) exist in different niches within the epidermis and maintain discrete epidermal compartments, but the exact contribution of each SC populations under physiological conditions is still unclear. In addition, the precise mechanisms controlling the balance between proliferation and differentiation of epidermal SC still remain elusive. Recent studies provide new insights into these important questions by showing the contribution of hair follicle SC to the sebaceous lineage and the importance of chromatin modifications and micro-RNAs (miRs) in regulating epidermal SCs renewal and differentiation. In this review, we will discuss the importance of these papers to our understanding of the mechanisms that control epidermal SC functions.     

Designer skin: lineage commitment in postnatal epidermis

The epidermis is populated by stem cells that produce daughters that differentiate to form the interfollicular epidermis, hair follicles and sebaceous glands. Diffusible factors, cell-cell contact and extracellular matrix proteins are all important components of the microenvironment of individual stem cells and profoundly affect the differentiation pathways selected by their progeny. Here, we summarize what is known about stem-cell populations and lineage relationships within the epidermis. We also present evidence that postnatal epidermis can be reprogrammed, altering the number and location of cells that differentiate along specific epidermal lineages.