Rac1 is crucial for hair follicle integrity but is not essential for maintenance of the epidermis

Rac1 is a small GTPase that regulates the actin cytoskeleton but also other cellular processes. To investigate the function of Rac1 in skin, we generated mice with a keratinocyte-restricted deletion of the rac1 gene. Rac1-deficient mice lost nearly all of their hair within a few weeks after birth. The nonpermanent part of mutant hair follicles developed constrictions; lost expression of hair follicle-specific keratins, E-cadherin, and alpha6 integrin; and was eventually removed by macrophages. The permanent part of hair follicles and the sebaceous glands were maintained, but no regrowth of full-length hair follicles was observed. In the skin of mutant mice, epidermal keratinocytes showed normal differentiation, proliferation, cell-cell contacts, and basement membrane deposition, demonstrating no obvious defects of Rac1-deficient epidermis in vivo. In vitro, Rac1-null keratinocytes displayed a strong spreading defect and slightly impaired adhesion. These data show that Rac1 plays an important role in sustaining the integrity of the lower part of hair follicles but not in maintenance of the epidermis.     

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.     

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.     

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.     

Cidea Control of Lipid Storage and Secretion in Mouse and Human Sebaceous Glands

Sebaceous glands are skin appendages that secrete sebum onto hair follicles to lubricate the hair and maintain skin homeostasis. In this study, we demonstrated that Cidea is expressed at high levels in lipid-laden mature sebocytes and that Cidea deficiency led to dry hair and hair loss in aged mice. In addition, Cidea-deficient mice had markedly reduced levels of skin surface lipids, including triacylglycerides (TAGs) and wax diesters (WDEs), and these mice were defective in water repulsion and thermoregulation. Furthermore, we observed that Cidea-deficient sebocytes accumulated a large number of smaller-sized lipid droplets (LDs), whereas overexpression of Cidea in human SZ95 sebocytes resulted in increased lipid storage and the accumulation of large LDs. Importantly, Cidea was highly expressed in human sebaceous glands, and its expression levels were positively correlated with human sebum secretion. Our data revealed that Cidea is a crucial regulator of sebaceous gland lipid storage and sebum lipid secretion in mammals and humans.     

Phospholipase C-delta1 and -delta3 are essential in the trophoblast for placental development

Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in phosphoinositide turnover and is involved in a variety of physiological functions. We analyzed PLCdelta1 knockout mice and found that PLCdelta1 is required for the maintenance of skin homeostasis. However, there were no remarkable abnormalities except hair loss and runting in PLCdelta1 knockout mice, even though PLCdelta1 is broadly distributed. Here, we report that mice lacking both PLCdelta1 and PLCdelta3 died at embryonic day 11.5 (E11.5) to E13.5. PLCdelta1/PLCdelta3 double-knockout mice exhibited severe disruption of the normal labyrinth architecture in the placenta and decreased placental vascularization, as well as abnormal proliferation and apoptosis of trophoblasts in the labyrinth area. Furthermore, PLCdelta1/PLCdelta3 double-knockout embryos supplied with a normal placenta by the tetraploid aggregation method survived beyond E14.5, clearly indicating that the embryonic lethality is caused by a defect in trophoblasts. On the basis of these results, we conclude that PLCdelta1 and PLCdelta3 are essential in trophoblasts for placental development.     

Expression of DeltaNLef1 in mouse epidermis results in differentiation of hair follicles into squamous epidermal cysts and formation of skin tumours.

To examine the consequences of repressing beta-catenin/Lef1 signalling in mouse epidermis, we expressed a DeltaNLef1 transgene, which lacks the beta-catenin binding site, under the control of the keratin 14 promoter. No skin abnormalities were detected before the first postnatal hair cycle. However, from 6 weeks of age, mice underwent progressive hair loss which correlated with the development of dermal cysts. The cysts were derived from the base of the hair follicles and expressed morphological and molecular markers of interfollicular epidermis. Adult mice developed spontaneous skin tumours, most of which exhibited sebaceous differentiation, which could be indicative of an origin in the upper part of the hair follicle. The transgene continued to be expressed in the tumours and beta-catenin signalling was still inhibited, as evidenced by absence of cyclin D1 expression. However, patched mRNA expression was upregulated, suggesting that the sonic hedgehog pathway might play a role in tumour formation. Based on our results and previous data on the consequences of activating beta-catenin/Lef1 signalling in postnatal keratinocytes, we conclude that the level of beta-catenin signalling determines whether keratinocytes differentiate into hair or interfollicular epidermis, and that perturbation of the pathway by overexpression of DeltaNLef1 can lead to skin tumour formation.     

Beta-catenin and Hedgehog signal strength can specify number and location of hair follicles in adult epidermis without recruitment of bulge stem cells

Using K14deltaNbeta-cateninER transgenic mice, we show that short-term, low-level beta-catenin activation stimulates de novo hair follicle formation from sebaceous glands and interfollicular epidermis, while only sustained, high-level activation induces new follicles from preexisting follicles. The Hedgehog pathway is upregulated by beta-catenin activation, and inhibition of Hedgehog signaling converts the low beta-catenin phenotype to wild-type epidermis and the high phenotype to low. beta-catenin-induced follicles contain clonogenic keratinocytes that express bulge markers; the follicles induce dermal papillae and provide a niche for melanocytes, and they undergo 4OHT-dependent cycles of growth and regression. New follicles induced in interfollicular epidermis are derived from that cellular compartment and not through bulge stem cell migration or division. These results demonstrate the remarkable capacity of adult epidermis to be reprogrammed by titrating beta-catenin and Hedgehog signal strength and establish that cells from interfollicular epidermis can acquire certain characteristics of bulge stem cells.     

Hair follicular expression and function of group X secreted phospholipase A2 in mouse skin

Although perturbed lipid metabolism can often lead to skin abnormality, the role of phospholipase A(2) (PLA(2)) in skin homeostasis is poorly understood. In the present study we found that group X-secreted PLA(2) (sPLA(2)-X) was expressed in the outermost epithelium of hair follicles in synchrony with the anagen phase of hair cycling. Transgenic mice overexpressing sPLA(2)-X (PLA2G10-Tg) displayed alopecia, which was accompanied by hair follicle distortion with reduced expression of genes related to hair development, during a postnatal hair cycle. Additionally, the epidermis and sebaceous glands of PLA2G10-Tg skin were hyperplasic. Proteolytic activation of sPLA(2)-X in PLA2G10-Tg skin was accompanied by preferential hydrolysis of phosphatidylethanolamine species with polyunsaturated fatty acids as well as elevated production of some if not all eicosanoids. Importantly, the skin of Pla2g10-deficient mice had abnormal hair follicles with noticeable reduction in a subset of hair genes, a hypoplasic outer root sheath, a reduced number of melanin granules, and unexpected up-regulation of prostanoid synthesis. Collectively, our study highlights the spatiotemporal expression of sPLA(2)-X in hair follicles, the presence of skin-specific machinery leading to sPLA(2)-X activation, a functional link of sPLA(2)-X with hair follicle homeostasis, and compartmentalization of the prostanoid pathway in hair follicles and epidermis.     

Dynamic regulation of retinoic acid-binding proteins in developing, adult and neoplastic skin reveals roles for beta-catenin and Notch signalling

Retinoic acid (RA) signalling is essential for epidermal differentiation; however, the mechanisms by which it acts are largely unexplored. Partitioning of RA between different nuclear receptors is regulated by RA-binding proteins. We show that cellular RA-binding proteins CRABP1 and CRABP2 and the fatty acid-binding protein FABP5 are dynamically expressed during skin development and in adult tissue. CRABP1 is expressed in embryonic dermis and in the stroma of skin tumours, but confined to the hair follicle dermal papilla in normal postnatal skin. CRABP2 and FABP5 are expressed in the differentiating cells of sebaceous gland, interfollicular epidermis and hair follicles, with FABP5 being a prominent marker of sebaceous glands and anagen follicle bulbs. All three proteins are upregulated in response to RA treatment or Notch activation and are negatively regulated by Wnt/β-catenin signalling. Ectopic follicles induced by β-catenin arise from areas of the sebaceous gland that have lost CRABP2 and FABP5; conversely, inhibition of hair follicle formation by N-terminally truncated Lef1 results in upregulation of CRABP2 and FABP5. Our findings demonstrate that there is dynamic regulation of RA signalling in different regions of the skin and provide evidence for interactions between the RA, β-catenin and Notch pathways.     

c-Myc activation in transgenic mouse epidermis results in mobilization of stem cells and differentiation of their progeny

BACKGROUND: The epidermis is maintained throughout adult life by pluripotential stem cells that give rise, via daughter cells of restricted self-renewal capacity and high differentiation probability (transit-amplifying cells), to interfollicular epidermis, hair follicles, and sebaceous glands. In vivo, transit-amplifying cells are actively cycling, whereas stem cells divide infrequently. Experiments with cultured human keratinocytes suggest that c-Myc promotes epidermal-stem cell differentiation. However, Myc is a potent oncogene that suppresses differentiation and causes reversible neoplasia when expressed in the differentiating epidermal layers of transgenic mice. To investigate the effects of c-Myc on the stem cell compartment in vivo, we targetted c-MycER to the basal layer of transgenic mouse epidermis. RESULTS: The activation of c-Myc by the application of 4-hydroxy-tamoxifen caused progressive and irreversible changes in adult epidermis. Proliferation was stimulated, but interfollicular keratinocytes still underwent normal terminal differentiation. Hair follicles were abnormal, and sebaceous differentiation was stimulated at the expense of hair differentiation. The activation of c-Myc by a single application of 4-hydroxy-tamoxifen was as effective as continuous treatment in stimulating proliferation and sebocyte differentiation, and the c-Myc-induced phenotype continued to develop even after the grafting of treated skin to an untreated recipient. CONCLUSIONS: We propose that transient activation of c-Myc drives keratinocytes from the stem to the transit-amplifying compartment and thereby stimulates proliferation and differentiation along the epidermal and sebaceous lineages. The ability, demonstrated here for the first time, to manipulate exit from the stem cell compartment in vivo will facilitate further investigations of the relationship between stem cells and cancer.     

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.     

皮肤表皮干细胞的研究进展

成体的皮肤一生都在不断的自我更新,其中的毛囊还是保证毛发进行生长-脱落周期循环的细胞组织学基础。存在于表皮内的干细胞维持了成体皮肤的自我平衡及毛发再生。表皮是由构体分子组成。每个构体分子包含毛皮脂单位（毛囊和皮脂腺）及其周围的毛囊间表皮。毛囊间表皮具有祖细胞,损伤时能自我更新;毛囊具有多能干细胞,在新毛发周期开始或者损伤时能够启动干细胞功能,为毛囊的生长或表皮的修复提供细胞来源。本文概述了当前对表皮干细胞的认识,着重阐明毛囊间表皮内有祖细胞的证据,毛囊间表皮干细胞在体外的自我更新能力,毛囊膨突部内干细胞的特征和一些相关基因的表达等。     

Abnormal epidermal differentiation and impaired epithelial-mesenchymal tissue interactions in mice lacking the retinoblastoma relatives p107 and p130

The functions of p107 and p130, members of the retinoblastoma family, include the control of cell cycle progression and differentiation in several tissues. Our previous studies suggested a role for p107 and p130 in keratinocyte differentiation in vitro. We now extend these data using knockout animal models. We found impaired terminal differentiation in the interfollicular keratinocytes of p107/p130-double-null mice epidermis. In addition, we observed a decreased number of hair follicles and a clear developmental delay in hair, whiskers and tooth germs. Skin grafts of p107/p130-deficient epidermis onto NOD/scid mice showed altered differentiation and hyperproliferation of the interfollicular keratinocytes, thus demonstrating that the absence of p107 and p130 results in the deficient control of differentiation in keratinocytes in a cell-autonomous manner. Besides normal hair formation, follicular cysts, misoriented and dysplastic follicles, together with aberrant hair cycling, were also observed in the p107/p130 skin transplants. Finally, the hair abnormalities in p107/p130-null skin were associated with altered Bmp4-dependent signaling including decreased DeltaNp63 expression. These results indicate an essential role for p107 and p130 in the epithelial-mesenchimal interactions.     

SD大鼠皮肤组织病理学观察

目的观察不同日龄SD大鼠皮肤组织学结构。方法10％甲醛固定,行石蜡切片,HE染色。结果新生大鼠皮肤较薄,透明层缺乏,皮脂腺发育良好。6月龄时表皮、真皮和皮下组织明显增厚,毛囊增粗,生长旺盛,毛囊深入皮下脂肪层。24月龄时,大鼠皮脂腺及汗腺萎缩,表皮变薄,真皮成纤维细胞、血管数量减少,弹力纤维变细。结论不同日龄SD大鼠皮肤组织学结构有差异。     

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.     

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.     

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.