A conditional transgenic mouse line for targeted expression of the stem cell marker LGR5.

LGR5 is a known marker of embryonic and adult stem cells in several tissues. In a mouse model, Lgr5+ cells have shown tumour-initiating properties, while in human cancers, such as basal cell carcinoma and colon cancer, LGR5 expression levels are increased: however, the effect of increased LGR5 expression is not fully understood. To study the effects of elevated LGR5 expression levels we generated a novel tetracycline-responsive, conditional transgenic mouse line expressing human LGR5, designated TRELGR5. In this transgenic line, LGR5 expression can be induced in any tissue depending on the expression pattern of the chosen transcriptional regulator. For the current study, we used transgenic mice with a tetracycline-regulated transcriptional transactivator linked to the bovine keratin 5 promoter (K5tTA) to drive expression of LGR5 in the epidermis. As expected, expression of human LGR5 was induced in the skin of double transgenic mice (K5tTA;TRELGR5). Inducing LGR5 expression during embryogenesis and early development resulted in macroscopically and microscopically detectable phenotypic changes, including kink tail, sparse fur coat and enlarged sebaceous glands. The fur and sebaceous gland phenotypes were reversible upon discontinued expression of transgenic LGR5, but this was not observed for the kink tail phenotype. There were no apparent phenotypic changes if LGR5 expression was induced at three weeks of age. The results demonstrate that increased expression of LGR5 during embryogenesis and the neonatal period alter skin development and homeostasis.

Overexpression of epigen during embryonic development induces reversible, epidermal growth factor receptor-dependent sebaceous gland hyperplasia.

The epidermal growth factor receptor (EGFR) system is a key regulator of epithelial development and homeostasis. Its functions in the sebaceous gland (SG), however, remain poorly characterized. In this study, using a transgenic mouse line with tissue-specific and inducible expression of the EGFR ligand epigen, we showed that increased activation of the EGFR in skin keratinocytes results in enlarged SGs and increased sebum production. The phenotype can be reverted by interrupting transgene expression and is EGFR dependent, as gland size and sebum levels return to normal values after crossing to the EGFR-impaired mouse line Wa5. Intriguingly, however, the SG enlargement appears only if EGFR activation occurs before birth. Importantly, the enlarged sebaceous glands are associated with an increased expression of the transcription factor MYC and of the transmembrane proteins LRIG1, an established negative-feedback regulator of the EGFR/ERBB tyrosine kinase receptors and a stem cell marker. Our findings identify EGFR signaling as a major pathway determining SG activity and suggest a functional relationship between the EGFR/ERBB system and MYC/LRIG1 in the commitment of stem cells toward specific progenitor cell types, with implications for our understanding of their role in tissue development, homeostasis, and disease.

Expression of caspase-14 and keratin-19 in the human epidermis and appendages during fetal skin development.

Caspase-14 is a seemingly non-apoptotic caspase involved in keratinocyte differentiation and cornification of the skin. Keratin-19 is an epithelial marker and a potential marker of epidermal stem cells that is expressed during human fetal skin development. We examined the immunohistochemical expression of caspase-14 in relation to CK-19 in the human fetal skin during development and perinatally, to assess their role in human skin maturation. Skin samples were received at autopsy. In the fetal epidermis, caspase-14 was predominantly expressed in the more differentiated layers, gradually disappearing from the basal layer toward term. By contrast, keratin-19 expression gradually decreased with epidermal maturation through gestation (rho = -0.949; p = 0.0001) and was a marker of the germinative layers. Keratin-19 was preserved in scarce basal cell nests at term and postnatally. Caspase-14 and keratin-19 were inversely expressed in the differentiating epidermal layers through gestation (p < 0.0001). Concerning the appendages, in hair follicles and sebaceous glands, caspase-14 located preferentially in the more differentiated layers of the inner root sheath, whereas keratin-19 was expressed in the outer sheath. Eccrine sweat glands showed a variable pattern of caspase-14 and keratin-19 expression. In conclusion, caspase-14 emerged as a marker of human skin differentiation during development, while keratin-19 marked the germinative epithelial layers in the fetal epidermis and appendages and possibly the nests of epidermal stem cells.

Stem cell dynamics in sebaceous gland morphogenesis in mouse skin.

The hair follicle (HF) and the sebaceous gland (SG) constitute the two integral parts of the pilosebaceous unit and significantly contribute to the barrier function of mammalian skin. Considerable progress has been made in our understanding how HF formation is regulated. However, the development of the SG is poorly understood, both at the molecular and cellular level. Here, we investigate the process of SG morphogenesis and the dynamics of its cellular organisation in more detail. The spatial and temporal organisation of distinct stem and progenitor compartments was analysed during morphogenesis of the pilosebaceous unit in mouse tail epidermis. Our experiments reveal a dynamic expression pattern for diverse HF stem cell marker molecules including Sox9 and Lrig1. Surprisingly, Sox9 and Lrig1 are initially coexpressed by epidermal progenitor cells and are confined to different regions within the pilosebaceous unit when the specification of the sebocyte cell lineage takes place. We demonstrate that SG development at the distal part of the HF is driven by asymmetric cell fate decision of Lrig1 positive stem cells, whereas MTS24/Plet1 positive precursor cells seem not to play a role in this process. Importantly, our data clearly show that distinct stem and progenitor compartments are established at different time points of development. By studying the process of SG morphogenesis more precisely, we discovered that the two prominent SGs attached to one tail HF originate from one small cluster of sebocyte cells. Finally, we show regional specificity for HF patterning and spatio-temporal control of the underlying molecular signals initiating the development of the pilosebaceous unit.

[The sebaceous gland]. / Die Talgdrüse.

The development and function of the sebaceous gland in the fetal and neonatal periods appear to be regulated by maternal androgens and by endogenous steroid synthesis, as well as by other morphogens. The most apparent function of the glands is to excrete sebum. A strong increase in sebum excretion occurs a few hours after birth; this peaks during the first week and slowly subsides thereafter. A new rise takes place at about age 9 years with adrenarche and continues up to age 17 years, when the adult level is reached. The sebaceous gland is a target organ but also an important formation site of hormones, and especially of active androgens. Hormonal activity is based on an hormone (ligand)-receptor interaction, whereas sebocytes express a wide spectrum of hormone receptors. Androgens are well known for their effects on sebum excretion, whereas terminal sebocyte differentiation is assisted by peroxisome proliferator-activated receptor ligands. Estrogens, glucocorticoids, and prolactin also influence sebaceous gland function. In addition, stress-sensing cutaneous signals lead to the production and release of corticotrophin-releasing hormone from dermal nerves and sebocytes with subsequent dose-dependent regulation of sebaceous nonpolar lipids. Among other lipid fractions, sebaceous glands have been shown to synthesize considerable amounts of free fatty acids without exogenous influence. Atopic dermatitis, seborrheic dermatitis, psoriasis and acne vulgaris are some of the disease on which pathogenesis and severity sebaceous lipids may or are surely involved.

Blimp-1: a marker of terminal differentiation but not of sebocytic progenitor cells.

BACKGROUND: The role of stem cells in maintaining the sebaceous gland throughout the various stages of life is not satisfactorily resolved. In a recent article, the transcription factor B lymphocyte-induced maturation protein 1 (Blimp-1) was proposed as a marker of a population of unipotent progenitor cells that reside in the sebaceous gland, regulating its size and activity. METHODS: We used standard immunohistochemical methods to examine Blimp-1 expression in samples from embryonic, fetal and adult human skin and in 119 sebaceous lesions comprising all major categories of sebocytic lineage, including hamartomas, cysts and benign and malignant neoplasms. RESULTS: Blimp-1 is expressed late in embryonic development and is restricted to the evolving sebaceous gland, the terminally differentiating components of the hair follicle and nail organ and the granular layer. This pattern is preserved into adult life. In all sebaceous lesions, Blimp-1 labels only the most mature cellular constituents. CONCLUSIONS: The reported expression pattern is difficult to reconcile with a function of Blimp-1 as a marker for sebocytic progenitor cells but indicates a major role in terminal differentiation. Within the interfollicular epidermis, its exclusive localization to the granular layer suggests a central function in skin barrier homeostasis in the human.

Thematic review series: skin lipids. Sebaceous gland lipids: friend or foe?

Sebaceous glands are intriguing glands that are found throughout the human body except on the palms of the hands and soles of the feet. The true function of these glands has yet to be determined, but there are several theories, including antioxidant effects, antibacterial effects, and transport of pheromones. Sebaceous glands produce lipids that are involved in the pathogenesis of one of the most prevalent diseases of adolescence, acne. Although the majority of lipids produced by the sebaceous gland are also produced in other areas of the body, there are two that are characteristic of the sebaceous gland, wax esters and squalene. This review seeks to present an update on the physiology of the sebaceous glands, with particular emphasis on the production of sebaceous lipids.

Blimp1 defines a progenitor population that governs cellular input to the sebaceous gland.

Epidermal lineage commitment occurs when multipotent stem cells are specified to three lineages: the epidermis, the hair follicle, and the sebaceous gland (SG). How and when a lineage becomes specified remains unknown. Here, we report the existence of a population of unipotent progenitor cells that reside in the SG and express the transcriptional repressor Blimp1. Using cell-culture studies and genetic lineage tracing, we demonstrate that Blimp1-expressing cells are upstream from other cells of the SG lineage. Blimp1 appears to govern cellular input into the gland since its loss leads to elevated c-myc expression, augmented cell proliferation, and SG hyperplasia. Finally, BrdU labeling experiments demonstrate that the SG defects associated with loss of Blimp1 lead to enhanced bulge stem cell activity, suggesting that when normal SG homeostasis is perturbed, multipotent stem cells in the bulge can be mobilized to correct this imbalance.

Characterization of Lef-1 promoter segments that facilitate inductive developmental expression in skin.

Lymphoid Enhancer Factor 1 (Lef-1) is an important developmental transcription factor required for the inductive formation of several epithelial-derived organs including hair follicles. Inductive expression of Lef-1 mRNA is tightly regulated during embryo development, suggesting the involvement of a highly regulated promoter. In vitro analysis of the Lef-1 gene has demonstrated the existence of at least two spatially distinct promoters with multiple transcriptional start sites that are responsive to the canonical Wnt/beta-catenin pathway. Regions of the Lef-1 promoter required for inductive regulation in vivo, however, have yet to be determined. To this end, we utilized LacZ-reporter transgenic mice to define segments of the human Lef-1 promoter capable of reproducing mesenchymal- or epithelial-restricted transcriptional patterns of Lef-1 expression during hair and vibrissa follicle development. These studies have revealed that a 110 bp Wnt/beta-catenin-responsive element, contained within a minimal 2.5 kb Lef-1 promoter, plays an important role in regulating mesenchymal, and potentially epithelial, expression during follicle development in mouse embryos. This 2.5 kb Lef-1 promoter also demonstrated inductive mesenchymal expression during postnatal anagen stage hair-follicle cycling. Additionally, analysis of Lef-1 promoter expression revealed previously uncharacterized regions of endogenous Lef-1 expression seen in the sebaceous glands of vibrissa and hair follicles in transgenic lines harboring the minimal Lef-1 promoter and additional intronic sequences. In summary, these studies have begun to dissect the transcriptional diversity of the human Lef-1 promoter during the hair/vibrissa follicle and sebaceous gland formation.

Dependence of fetal hairs and sebaceous glands on fetal adrenal cortex and possible control from adrenal medulla.

Human fetal adrenal development is characterized by rapid growth, high steroidogenic activity, and a distinct morphology, including a unique cortical compartment known as the fetal zone. For most of gestation, the predominant fetal zone accounts for 80-90% of the cortical volume and is the primary site of growth and steroidogenesis, producing 100-200 mg/day of the androgenic steroid, dehydroepiandrosterone sulfate (DHEA-S). The physiological role of this zone during intrauterine life is not well understood. While the glands appear to be capable of DHEA-S synthesis early in gestation (8-10 weeks), we noticed that this event precedes the differentiation of hairs and sebaceous glands. Hairs begin to develop between 9 and 12 weeks and sebaceous glands between 13 and 15 weeks of gestation. Sebaceous glands form an oily secretion - sebum that mixes with desquamated epidermal cells to form vernix caseosa. Vernix caseosa protects the developing skin from constant exposure to amniotic fluid, and hairs helps to hold the vernix caseosa on the skin. We suggest therefore that the human fetal adrenal cortex produces DHEA-S beginning at around 8-10 weeks of gestation in sufficient quantities to influence the growth of hairs and sebaceous glands. Soon after birth, the fetal zone atrophies, and adrenal androgen production decreases to minimal levels. As a consequence, in concordance with the rapid decrease in adrenal androgen levels and in consistent with our hypothesis, fetal hairs are shed and sebaceous glands shrink to small structures. The mechanism that regulates fetal adrenal androgen production is a key unanswered problem in human adrenal biology. Since there exists a close relationship between epinephrine and DHEA-S levels during adrenarche which shows modulatory interactions between adrenal androgen production and adrenomedullary function, we suggest again that adrenomedullary function might play a role in the control of fetal adrenal androgen secretion.

Role of hormones in pilosebaceous unit development.

Androgens are required for sexual hair and sebaceous gland development. However, pilosebaceous unit (PSU) growth and differentiation require the interaction of androgen with numerous other biological factors. The pattern of PSU responsiveness to androgen is determined in the embryo. Hair follicle growth involves close reciprocal epithelial-stromal interactions that recapitulate ontogeny; these interactions are necessary for optimal hair growth in culture. Peroxisome proliferator-activated receptors (PPARs) and retinoids have recently been found to specifically affect sebaceous cell growth and differentiation. Many other hormones such as GH, insulin-like growth factors, insulin, glucocorticoids, estrogen, and thyroid hormone play important roles in PSU growth and development. The biological and endocrinological basis of PSU development and the hormonal treatment of the PSU disorders hirsutism, acne vulgaris, and pattern alopecia are reviewed. Improved understanding of the multiplicity of factors involved in normal PSU growth and differentiation will be necessary to provide optimal treatment approaches for these disorders.

Essential role for Sonic hedgehog during hair follicle morphogenesis.

The hair follicle is a source of epithelial stem cells and site of origin for several types of skin tumors. Although it is clear that follicles arise by way of a series of inductive tissue interactions, identification of the signaling molecules driving this process remains a major challenge in skin biology. In this study we report an obligatory role for the secreted morphogen Sonic hedgehog (Shh) during hair follicle development. Hair germs comprising epidermal placodes and associated dermal condensates were detected in both control and Shh -/- embryos, but progression through subsequent stages of follicle development was blocked in mutant skin. The expression of Gli1 and Ptc1 was reduced in Shh -/- dermal condensates and they failed to evolve into hair follicle papillae, suggesting that the adjacent mesenchyme is a critical target for placode-derived Shh. Despite the profound inhibition of hair follicle morphogenesis, late-stage follicle differentiation markers were detected in Shh -/- skin grafts, as well as cultured vibrissa explants treated with cyclopamine to block Shh signaling. Our findings reveal an essential role for Shh during hair follicle morphogenesis, where it is required for normal advancement beyond the hair germ stage of development.

Expression of the small heat shock protein HSP 27 in developing human skin.

The 27 kDa heat shock protein (HSP 27) is expressed in keratinocytes of the upper epidermal layers, and recent evidence suggests that this protein is involved in the regulation of epidermal differentiation. The expression of HSP 27 was investigated in developing human skin by immunohistochemistry utilizing a specific monoclonal antibody. We used formalin-fixed, paraffin-embedded tissue of abdominal skin obtained from 34 human fetuses ranging between 13 and 30 weeks estimated gestational age (EGA). We found that HSP 27 is not expressed in keratinocytes until week 14 EGA. At this stage staining is observed in the periderm and the upper intermediate cells but not in hair germs. During further development, HSP 27 expression correlates with increasing epidermal differentiation, i.e. shedding of the periderm and beginning of keratinization. HSP 27 expression is confined to the upper cell layers and sparse basal cells. In hair follicles, HSP 27 can be detected in the innermost cell layer of the outer root sheath and in keratinocytes of the bulge identical to what is observed in adult skin. The hair papilla, matrix cells and sebaceous glands are negative for HSP 27 and remain so during further development. In eccrine sweat glands of the 24th week EGA, HSP 27 is confined to the superficial cell layer of the sweat ducts. In the present report we demonstrate differentiation-related expression of HSP 27 in developing human skin. Further in vitro studies will address the molecular function of HSP 27 in epidermal differentiation and development.

HOX homeobox genes exhibit spatial and temporal changes in expression during human skin development.

The spatial and temporal deployment of HOX homeobox genes along the spinal axis and in limb buds during fetal development is a key program in embryonic pattern formation. Although we have previously reported that several of the HOX homeobox genes are expressed during murine skin development, there is no information about developmental expression of HOX genes in human skin. We have now used reverse transcriptase polymerase chain reaction, in conjunction with a set of degenerate oligonucleotide primers, to identify a subset of HOX genes that are expressed during human fetal skin development. In situ hybridization analyses demonstrated that there were temporal and spatial shifts in expression of these genes. Strong HOXA4 expression was detected in the basal cell layers of 10 wk fetal epidermis and throughout the epidermis and dermis of 17 wk skin, whereas weak signal was present in the granular layer of newborn and adult skin. The expression patterns of HOXA5 and HOXA7 were similar, but their expression was weaker. In situ hybridization analysis also revealed strong HOXC4 and weaker HOXB7 expression throughout fetal development, whereas HOXB4 was expressed at barely detectable levels. Differential HOX gene expression was also observed in developing hair follicles, and sebaceous and sweat glands. None of the HOX genes examined were detected in the adult dermis.

Clear cell hidradenoma associated with the folliculo-sebaceous-apocrine unit. Histologic study of five cases.

Hidradenomas are benign adnexal neoplasms of uncertain derivation. Although most investigators traditionally have considered these neoplasms to exhibit eccrine differentiation, with only occasional reports supporting apocrine derivation, subdivision of hidradenomas into two groups was recently suggested: those with eccrine differentiation (or poroid hidradenomas) and those exhibiting apocrine differentiation (or clear cell hidradenomas). We have observed five cases of clear cell hidradenomas manifesting unquestionable apocrine characteristics excised from sites other than the axilla, and displaying continuity with the epidermis through follicles. Because of the common embryologic derivation of apocrine glands, sebaceous glands, and hair follicles, and their histologic integration as the folliculo-sebaceous-apocrine unit, these findings suggest a possible origin of certain apocrine hidradenomas from pluripotential germinative cells present in follicles.

Epidermal growth factor inhibits morphogenesis of the embryonic quail uropygial gland cultured in vitro.

Formation of the uropygial papilla and glandular lumena was inhibited when the uropygial rudiment of a day 8 1/3 quail embryo was cultured for 2 days in a chemically defined medium in the presence of 50 ng/mL of epidermal growth factor (EGF). The epithelium of EGF-treated explants remained at the placode stage, or underwent minor invagination into the mesenchyme and became stratified like that of a 12- or 13-day-old embryo. EGF promoted cellular proliferation in the uropygial epithelium and the epidermis adjacent to the gland and it shortened the lag phase of proliferation and markedly stimulated epithelial DNA synthesis, detected immunocytochemically by labeling explants with 5-bromodeoxyuridine (BrdU). The maximal labeling index in EGF-treated uropygial epithelium was 55% higher than in the control. Electron microscopic observation revealed that the basal lamina had become irregular in the EGF-treated explants and that epithelial cytoplasmic processes penetrated through the basal lamina toward the mesenchyme. These same phenomena are observed in vivo when the glandular buds are formed during day 12-13. Some precocious changes occurred in the uropygial epithelium when the rudiment was cultured in the presence of EGF.

Role of androgens in the developmental biology of the pilosebaceous unit.

The growth and development of pilosebaceous units in their characteristic pattern depends on the interaction of androgens and diverse biologic factors. Stromal-epithelial interactions are essential features. Considerable evidence suggests that androgens stimulate the growth of sensitive pilosebaceous units primarily by acting on specific stromal cells and that androgens and retinoic acid interact to regulate specific stages of sebocyte differentiation.

Histogenetic potential of rat hind-limb interdigital tissues prior to and during the onset of programmed cell death.

The histogenetic potential of interdigital tissues isolated from the autopod of rat embryonic hind-limbs between 14.5 and 16.5 days was investigated. A wedge of tissue containing ectoderm and mesoderm was excised from between the developing digits and grafted beneath the kidney capsule of adult rats for two weeks. We have previously demonstrated that the renal capsule is an excellent site for permitting limb tissues to proliferate and differentiate (Chan et al.: J. Exp. Zool., 260:74-83, 1991). At 14.5 days, when cell death (revealed with neutral red stains) within the interdigital zone was limited to the apical ectodermal ridge (AER), the interdigital mesoderm was capable of developing into bone, cartilage, and loose connective tissue in the kidney. It was estimated that the skeletal elements occupied approximately 38% of the overall area of the grafts. In addition, the ectoderm was able to produce keratinized epithelium, hair follicles, and sebaceous glands. In 15.5 day autopod, necrosis was present both in the AER and the mesoderm between the AER and marginal sinus. Interdigital mesoderm obtained from this stage of development formed cartilage but not as extensively as that derived from 14.5 day autopod (4% as compared with 38%). Necrotic cells were present in all of the interdigital zones at 16.5 days. Ten explants were introduced into the kidney at this stage, but only 4 grafts were recovered after 2 weeks. In all cases, the explants did not produce cartilage. Only a small amount of keratinized epithelium and loose connective tissue was found. In summary the interdigital mesoderm has the potential to develop bone, cartilage, and loose connective tissue, but this ability is progressively lost during morphogenesis.

Embryology of the hair follicle.

A study of hair follicle development has enabled us to differentiate four stages by relating them to corresponding gestation ages. Stage I of the hair bud is characterized by an epiblastic proliferation penetrating the subjacent mesenchyma at the extremity of which mesenchymal cells accumulate. This appears, at the level of the lower lip, before the 11th week of intrauterine life. Stage II of the hair bulb is reached on the 12th-13th week of intrauterine life, as soon as the distal extremity of the hair bud, which has extended, becomes depressed at the mesenchymal papilla level. Stage III is defined by the observation of hair cone and rough sebaceous glands on the 15th week of intrauterine life. Finally, stage IV shows a differentiated sebaceous gland with a hair which comes through the skin surface on the 18th week of intrauterine life.

Differential keratin gene expression during the differentiation of the cement gland of Xenopus laevis.

The expression of both epidermal and nonepidermal keratins has been detected in the cement gland of Xenopus laevis by antibody staining. Northern blot and in situ hybridizations with gene-specific probes indicated the expression of the nonepidermal keratin, XK endo B, and the embryonic epidermal keratin, XK70, in the cement gland. Furthermore, since explanted animal pole cells can be induced to differentiate into cement gland cells in vitro by incubation in NH4Cl, we have demonstrated the in vitro induction of XK endo B, maintenance of XK70, and repression of another embryonic epidermal keratin, XK81. This is the first report of keratin gene expression in the cement gland.