Involucrin, but not filaggrin and Kdap mRNA, expression is downregulated in 3-D cultures of intact rat hair bulbs after calcium stimulation

The hair follicle consists of several distinctive epidermal cell layers. The hair root, which undergoes keratinization, is surrounded by two sheaths: the inner root sheath (IRS) and the outer root sheath (ORS). The ORS is continuous with the basal layer of the epidermis. Its cells do not keratinize in situ, unlike IRS. We have previously demonstrated that keratinization of the ORS was prevented by contact with the IRS in hair follicle mid-segments (i.e. fragments dissected from skin at the level above the hair bulb and below the opening of the sebaceous gland duct) cultured on agarose layer. The purpose of this study was to determine whether the same applies to the hair bulb. After isolation, intact bulbs or hair bulb-derived cells were incubated in suspension in a low or high calcium medium. The level of mRNA for differentiation markers: involucrin, filaggrin, keratinocyte differentiation associated protein and trichohyalin, was studied by RealTime PCR. We observed increased Ca(2+) upregulated expression of involucrin, filaggrin, trichohyalin and Kdap in cultures of bulb-derived cells, but in hair bulbs downregulation of involucrin and trichohyalin was observed. We concluded that the inner root sheath exerts an inhibitory effect on the expression of involucrin and trichohyalin already in the hair bulbs. The observation that downregulation of involucrin expression under Ca(2+) influence occurs both in hair bulb and midsegments could simplify future experiments, since their separation does not seem to be necessary.     

Sox9 is essential for outer root sheath differentiation and the formation of the hair stem cell compartment

BACKGROUND: The mammalian hair represents an unparalleled model system to understand both developmental processes and stem cell biology. The hair follicle consists of several concentric epithelial sheaths with the outer root sheath (ORS) forming the outermost layer. Functionally, the ORS has been implicated in the migration of hair stem cells from the stem cell niche toward the hair bulb. However, factors required for the differentiation of this critical cell lineage remain to be identified. Here, we describe an unexpected role of the HMG-box-containing gene Sox9 in hair development. RESULTS: Sox9 expression can be first detected in the epithelial component of the hair placode but then becomes restricted to the outer root sheath (ORS) and the hair stem cell compartment (bulge). Using tissue-specific inactivation of Sox9, we demonstrate that this gene serves a crucial role in hair differentiation and that skin deleted for Sox9 lacks external hair. Strikingly, the ORS acquires epidermal characteristics with ectopic expression of GATA3. Moreover, Sox9 knock hair show severe proliferative defects and the stem cell niche never forms. Finally, we show that Sox9 expression depends on sonic hedgehog (Shh) signaling and demonstrate overexpression in skin tumors in mouse and man. CONCLUSIONS: We conclude that although Sox9 is dispensable for hair induction, it directs differentiation of the ORS and is required for the formation of the hair stem cell compartment. Our genetic analysis places Sox9 in a molecular cascade downstream of sonic hedgehog and suggests that this gene is involved in basal cell carcinoma.     

Cellular dynamics of the outer layers of the hair follicle of fine-wool sheep during the phase of stable hair growth

The structure, origin, and migration of outer sheath cells of the hair follicles of domestic sheep were studied by electron microscopic, autoradiographic, and histochemical (glycogen) methods in order to understand the role of this layer in hair morphogenesis. We demonstrated that the cells of the outer layers of the outer sheath interpose into the inner “companion” layer of the outer sheath. Although this process takes place all along the hair follicle from the lower bulb up to the sebaceous glands orifices, it mainly takes place over the bulb. Labeled cells interposed into the companion layer reach sebaceous glands orifices more than 24 h faster than labeled cells of the inner sheath and hair, because these cells included the label not in the bulb cambium (as hair and inner sheath) but over the bulb, and from this point they start movement. Interposition of cells into the companion layer must cause increase of its volume and additional volume supposed to be led away into the pillar canal around the hair near the sebaceous glands orifices. This can provide the mechanism of the hair and inner sheath promotion to sebaceous gland orifices.     

Expression of galectin-1 and -3 and of accessible binding sites during murine hair cycle

Although protein-carbohydrate interactions are supposed to play key roles in cell adhesion, signalling and growth control. Their exact role in skin physiology has only recently been investigated. The endogenous lectins galectin-1 and galectin-3 have been identified in skin including hair follicles. Here, we analyzed the expression and distribution of these galectins and their binding sites in C57BL/6 mice during hair cycle. The expression of galectin-1 and galectin-3 binding sites was found to be predominantly hair cycle-dependent showing some overlapping to the expression of galectin-1 and -3. The outer root sheath (ORS) expressed galectin-1 binding sites during anagen IV to VI and in early catagen, whereas galectin-1 was expressed from early anagen to late catagen. The ORS expressed galectin-3 binding sites during catagen transition corresponding to a galectin-3 expression during anagen V and catagen. The innermost layer of the ORS expressed galectin-3 binding sites during anagen VI until catagen VIII, but galectin-3 during anagen III to IV and catagen. The inner root sheath (IRS) expressed galectin-3 binding sites only in anagen IV but missed expression of any of the two galectins. The matrix cells expressed galectin-3 binding sites in catagen II-III as well as galectin-3 during anagen V to catagen IV. The present study provides the first evidence for a cycle-related expression of both galectin-1 and -3 and their binding sites during murine hair cycle.     

The postnatal development of the main olfactory bulb of the rat

The postnatal development from birth to 1 year of the main olfactory bulb was examined quantitatively. The volume of the main olfactory bulb increased over seven-fold by day 30 and remained unchanged thereafter. During the same period the volume of the granular layer increased 18-fold and the mean areas of the olfactory glomeruli increased seven-fold. The mean areas of mitral cell perikarya doubled between the neonatal and juvenile periods. The total number of the mitral cells, however, declined during the first three postnatal weeks. In the internal granular layer of the main olfactory bulb, 89% of the granule cells were acquired postnatally. Much of the cellular gain occurred during the first 3 weeks, with the period of maximum acquisition between days 8 and 14. The number of subependymal cells, the precursors of granule cells, reached a peak at 12 days and gradually declined. But some primitive cells could still be found at one year of age and there was an increase in the total number of granule cells beyond day 30. The mean nuber of internal granular layer cells in a single main olfactory bulb of adult rats was about 5 X 10(6); the number of mitral cells about 4 X 10(4). In the animals injected with 3H-thymidine on day 20 and killed 2 h after injection a small but significant proportion of cells was labelled in the subependymal layer but few in the internal granular layer. In the animals killed 20 and 40 days after injection there was a 10--11-fold rise in the proportion of labelled internal granular layer cells. The proportion of labelled internal granular layer cells decreased in longer survival groups but the total number of labelled cells remained the same, even in year-old animals. However, the total number of internal granular layer cells in the sections examined increased with age.     

Hair growth in mouse mutants affecting coat texture

The genetic control of hair growth has been studied in mice carrying the following coat texture genes: fz (fuzzy), soc (soft coat), hid (hair interior defect), sa (satin), It (lustrous), Ve (velvet), wa-1 (waved-1), Re (rex), Re ^wc (wavy coat) and pk (plucked).
A general effect on cells of epidermal origin, found in soc/soc and Ve /+ skin samples illustrates how common factors control developmental potential in both the stratum germinativum and the follicle bulb. A direct influence on follicle bulb development is also seen in fz/fz homozygotes in which the dermal papilla functions abnormally. The role of the bulb cells and the dermal papilla in the control of hair shaft calibre is discussed.
hid is a new gene, found in homozygous condition in all mice of the AKR inbred strain. hid and sa appear primarily to be concerned in the differentiation of the medulla.
In the hair waving mutants, waved-1, rex and wavy coat, the processes controlling hair movement within the follicle are disturbed. These genes appear to regulate internal root sheath function. When the normal relationship between internal root sheath and developing hair shaft is disturbed, shaft movement slows, with the subsequent development of shaft calibre abnormalities.
pk acts at the level of the sebaceous gland, disturbing the normal process of hair eruption. The roles of the internal root sheath, external root sheath and the sebaceous gland in hair eruption are discussed.
The abnormal epidermal layer in soc/soc and Ve /+ skin also disturbs hair eruption to a small extent. The resulting abnormalities this causes in hair shaft formation are compared with those found pk/pk samples and also with the similar effects of faulty hair movement in the hair waving mutants. An effect on pigmentation is also described.
The chemistry of keratinization appears to be normal in all these mutants.     

Hair cycle dynamics: the case of the human hair follicle

The existence of a growth and regeneration cycle makes the hair follicle a true paradigm of tissue homeostasis. Analysis of about 9000 cycles led us to propose a stochastic model of human hair dynamics. The existence of hair cycles implies that stem cells must be cyclically activated and hair melanin unit has to be renewed. Using different markers, we were able to identify two distinct epithelial stem cell reservoirs, located in the upper and lower thirds of the anagen hair follicle outer root sheath. These two reservoirs fuse during the regression phase and individualize again in the new forming anagen hair follicle. Using a set of antibodies specific of melanocyte lineage and melanogenesis, pigmentation unit turnover was followed throughout the entire hair cycle. In the terminal anagen hair, active melanocytes were localized on top of the dermal papilla, while amelanotic melanocytes were identified in the upper third of the outer root sheath (ORS). Those amelanotic melanocytes located in upper ORS probably represented a melanocyte reservoir for successive hair generation, since at the induction of anagen phase, some melanocytes were committed to cell division and melanogenesis was turned on, but only in the nascent hair bulb, close to the dermal papilla.     

Manipulation of outer root sheath cell survival perturbs the hair-growth cycle.

Transgenic mice that overexpress the anti-apoptotic gene bcl-xL under the control of the keratin 14 promoter have significantly shorter hair than non-transgenic littermates. The deficit in hair length correlated with a decrease in the duration of anagen, the growth phase of the hair cycle. A prolongation in telogen, the resting phase of the hair cycle, was also observed in adult animals. In the developing hair bulb, bcl-xL transgene expression was observed exclusively in the outer root sheath (ORS) cells. Bcl-xL expression enhanced the survival of ORS cells treated with apoptotic stimuli. The results suggest that preventing the apoptotic death of ORS cells during anagen leads to a more rapid termination of progenitor cell commitment/proliferation, while the increased survival of ORS cells during telogen delays the initiation of a new hair cycle. ORS cells produce fibroblast growth factor-5 (FGF-5), which acts in a paracrine fashion to terminate precursor cell division during anagen. The short hair phenotype of bcl-xL transgenic mice was substantially reversed in FGF-5-deficient mice. Thus, the production of growth inhibitory factors by ORS cells may provide a mechanism through which the hair-growth cycle is regulated by cell survival.     

Immunocytochemical localization of luteinizing hormone-releasing hormone within the olfactory bulb of pigs

LHRH was immunocytochemically localized within the olfactory bulb of prepubertal (n = 3), ovariectomized (n = 3), and hypophyseal-stalk-transected (HST) female pigs (n = 3). Perikarya of LHRH-immunoreactive neurons of all pigs were sparsely distributed mostly in the rostral half of the olfactory bulb, along the ventromedial and ventrolateral edge of the olfactory nerve layer, or at its interace with the glomerular layer. Processes from these cells and other LHRH containing axons either entered individual glomeruli forming a network within its interior or coursed around glomeruli penetrating into the external granular layers. Additional fibers penetrated into similar regions of the accessory olfactory bulb. Irregularly shaped perikarya were also detected within the internal granular layer of the ventral olfactory bulb, but only in tissue from HST pigs. From analysis of serial sections, there was no evidence of LHRH projections across the olfactory peduncle that connects the olfactory bulb with adjacent brain regions. If olfactory LHRH neurons are involved in reproductive behavior and physiology in the pig, this pathway involves additional unidentified intervening neurons. Endocrine factors probably influence the expression of immunoreactive LHRH in the internal granule layer, since their presence was revealed only in HST pigs.     

Histology and cytochemistry of human skin. XI. The distribution of beta-glucuronidase

1. Various amounts of beta-glucuronidase activity may be found in all of the cutaneous appendages. 2. In the epidermis, the basal layer and the Malpighian layer contain a moderate amount of it, but a band of cells, including the stratum granulosum and the cells immediately above it, is rich in beta-glucuronidase. 3. The cells of the duct of eccrine sweat glands have moderately strong enzyme activity, but those in the secretory coil are strongly reactive; small and large reactive granules are crowded in the reactive cytoplasm. 4. The cells of the secretory coil of the apocrine glands contain more beta-glucuronidase than any other cutaneous appendage. 5. In the sebaceous glands, a very strong concentration of enzyme activity is found in the undifferentiated peripheral cells, a smaller amount of it is found in the differentiating cells. 6. In active hair follicles, the largest amount of beta-glucuronidase is found in the outer root sheath and in the bulb. In the outer sheath, the strongest concentration is found around the level of the keratogenous zone of the cortex. The dermal papilla is strongly reactive. In quiescent hair follicles, the outer root sheath has a moderate amount of enzyme concentration, but the dermal papilla is unreactive. 7. In the dermis, the fibroblasts in the papillary layer, the smooth muscle cells of the arrectores pilorum and the tunica media of arteries, and the fat cells all exhibit enzyme activity. Mast cells show a great concentration of beta-glucuronidase.     

Expression of E6 and E7 papillomavirus oncogenes in the outer root sheath of hair follicles extends the growth phase and bypasses resting at telogen.

Hair follicle growth cycle proceeds through a series of stages in which strict control of cell proliferation, differentiation, and cell death occurs. Transgenic mice expressing human papillomavirus type 16 E6/E7 papillomavirus oncogenes in the outer root sheath (ORS) display a fur phenotype characterized by lower hair density and the ability to regenerate hair much faster than wild-type mice. Regenerating hair follicles of transgenic mice show a longer growth phase (anagen), and although bulb regression (catagen) occurs, rest at telogen was not observed. No abnormalities were detected during the first cycle of hair follicle growth, but by the second cycle, initiation of catagen was delayed, and rest at telogen was again not attained, even in the presence of estradiol, a telogen resting signal. In conclusion, expression of E6/E7 in the ORS delays entrance to catagen and makes cells of the ORS insensitive to telogen resting signals bearing to a continuous hair follicle cycling in transgenic mice.     

Histochemical localization of NADPH-diaphorase in the rat accessory olfactory bulb

The distribution of NADPH-diaphorase activity was examined inthe accessory olfactory bulb of the rat using a direct histochemicaltechnique. Labeled fibers and somata were found in all layersof the accessory olfactory bulb. The entire vomeronasal nerveand all vomeronasal glomeruli were strongly labeled, contraryto the main olfactory bulb, where only dorsomedial olfactoryglomeruli displayed NADPH-diaphorase activity. NADPH-diapborasepositive neurons were identified as periglomerular cells inthe glomerular layer and external plexiform layer, horizontalcells in the internal plexiform layer, and granule cells anddeep short-axon cells in the granule cell layer. The labeleddendrites of the granule cells formed a dense neuropile in thegranule cell layer, internal plexiform layer and external plexiformlayer. The staining pattern in the accessory olfactory bulbwas more complex than what has been previously reported, anddemonstrated both similarities and differences with the distributionof NADPH-diaphorase in the main olfactory bulb.     

HISTOLOGY AND CYTOCHEMISTRY OF HUMAN SKIN : XI. THE DISTRIBUTION OF β-GLUCURONIDASE

1. Various amounts of β-glucuronidase activity may be found in all of the cutaneous appendages. 2. In the epidermis, the basal layer and the Malpighian layer contain a moderate amount of it, but a band of cells, including the stratum granulosum and the cells immediately above it, is rich in β-glucuronidase. 3. The cells of the duct of eccrine sweat glands have moderately strong enzyme activity, but those in the secretory coil are strongly reactive; small and large reactive granules are crowded in the reactive cytoplasm. 4. The cells of the secretory coil of the apocrine glands contain more β-glucuronidase than any other cutaneous appendage. 5. In the sebaceous glands, a very strong concentration of enzyme activity is found in the undifferentiated peripheral cells, a smaller amount of it is found in the differentiating cells. 6. In active hair follicles, the largest amount of β-glucuronidase is found in the outer root sheath and in the bulb. In the outer sheath, the strongest concentration is found around the level of the keratogenous zone of the cortex. The dermal papilla is strongly reactive. In quiescent hair follicles, the outer root sheath has a moderate amount of enzyme concentration, but the dermal papilla is unreactive. 7. In the dermis, the fibroblasts in the papillary layer, the smooth muscle cells of the arrectores pilorum and the tunica media of arteries, and the fat cells all exhibit enzyme activity. Mast cells show a great concentration of β-glucuronidase.     

The electron histochemical demonstration of cystine-containing proteins in the guinea pig hair follicle

Summary A previously described electron histochemical method for detecting cystine in fully keratinised human hair has been modified for the examination of this amino acid residue amongst the cellular components of anagen guinea pig hair follicles. Using the technique the progressive incorporation of cystine into the hair cuticle and cortex has been observed. Although cystine was absent from the hair medulla and outer root sheath cells at all stages of development, a narrow layer of cystine-containing material was found adjacent to the cell membranes of hardened inner root sheath cells.     

Keratins of the human hair follicle: "Hyperproliferative''keratins consistently expressed in outer root sheath cells in vivo and in vitro

Keratins produced by morphologically distinct compartments of the human hair folicle (hHF) were analysed and compared to those produced by cultured hHF and interfollicular keratinocytes. Five of the major keratins, the basic keratins nos. 5 and 6 (apparent mol. mass 60 and 58 kDa) and the acidic keratins nos. 14, 16, and 17 (51, 49 and 48 kDa), could be labelled in intact hHF and were found in all fractions of the outer root sheath (ORS). The other major keratins, which were not labelled under these conditions (basic-neutral hHbI and -II; 60-62 kDa and acidic hHaI and -II; 40-42 kDa) were associated with hair shaft (hHS) both in the follicle and, virtually unchanged, in the distal part of the hair. Another, previously undescribed, group of proteins with keratin-like properties exhibiting a broad pI-spectrum (basic to slightly acidic: hIC-I, -II, -III, 64-67 kDa; distinctly acidic: hIC-IV, about 54 kDa) was detected in isolated inner root sheath (IRS), in the cuticular material shed from denuded hHS, and also in nail plates. In our experiments only ORS cells grew readily in culture irrespective of their origin from peripheral (mesenchyme-adjacent) or more central ORS-cell layers. In contrast to keratinocytes from interfollicular epidermis (IFE) the cultured ORS cells expressed a keratin set virtually identical to that expressed in vivo. This set also closely resembled that expressed by IFE keratinocyte cultures. The identity of the respective keratins (nos. 5, 6, 14, 16, and 17) present in all these cells in vivo and in vitro was confirmed by tryptic peptide mapping. The data indicated that the microenvironment (in situ) directs the differentiation of ORS cells in a manner comparable to the way it is directed by conventional culture conditions, with consistent expression of the "basal" and "hyperproliferative" set of keratins. This, however, does not exclude the possibility that other types of environmentally induced response may occur, as seen for example during the reepithelialization of superficial skin wounds by ORS cells.     

Dynamic change of histone H2AX phosphorylation independent of ATM and DNA-PK in mouse skin in situ

Histone H2AX undergoes phosphorylation on Ser 139 (γ-H2AX) rapidly in response to DNA double-strand breaks induced by exogenous stimuli, such as ionizing radiation. However, the endogenous phosphorylation pattern and modifier of H2AX remain unclear. Here we show that H2AX is regulated physically at the level of phosphorylation at Ser139 during a hair cycle in the mouse skin. In anagen hair follicles, γ-H2AX-positive cells were observed in the outer root sheath (ORS) and hair bulb in a cycling inferior region but not in a permanent superficial region. In telogen hair follicles, γ-H2AX-positive cells were only detected around the germ cell cap. In contrast, following X-irradiation, γ-H2AX was observed in various cell types including the ORS cells in the permanent superficial region. Furthermore, γ-H2AX-positive cells were detected in the skin of mice lacking either ATM or DNA-PK, suggesting that these kinases are not essential for phosphorylation in vivo.     

卷丹珠芽萌发的组织学观察

以卷丹(Lilium laneifolium)珠芽为试材,采用野外调查法、石蜡切片法、徒手切片法、离析法,观察珠芽各部分形态和结构,用分光光度法测定各片鳞叶的花青素和光合色素含量,为其珠芽繁殖生物学研究提供资料。结果表明,珠芽由鳞叶、鳞茎和不定根构成,鳞叶外表皮细胞具有发达的角质层,外表皮内侧1～2层叶肉细胞含有花青素;叶肉细胞含绿色造粉体,第1～3片鳞叶基部的绿色造粉体向不定根伸长方向集中分布,鳞叶色素含量由外至内逐渐降低;鳞叶维管束为外韧维管束。鳞茎主要由皮层和维管柱构成,鳞茎上端包括顶端分生组织和芽鞘,在下端细胞部分发生程序性死亡,但未发现类似叶片脱落时叶柄基部出现的离层结构。不定根起源于第2片鳞叶基部环生的鳞茎皮层细胞,不定根与周围鳞叶组织分离。在珠芽萌发过程中鳞叶的物质供给出现分化现象。     

Restoration of the epidermal phenotype by follicular outer root sheath cells in recombinant culture with dermal fibroblasts

In order to better understand how outer root sheath (ORS) cells are able to reepithelialize superficial skin wounds, the level of epidermal differentiation achieved by isolated ORS cells in vitro was determined. Using postmitotic human dermal fibroblasts (HDF) as efficient feeder cells, large numbers of ORS cells from individual follicles were generated. Passaged ORS cells were grown exposed to air on HDF-populated collagen gels in the CRD device (Noser and Limat, In vitro 23, 541-545, 1987) which allows histiotypic tissue organization. In such recombinant organotypic cultures, ORS cells developed distinct epidermal strata comparable to interfollicular keratinocytes (NEK). Ultrastructurally, desmosomes and intermediate filaments increased in number toward the epithelial surface and small keratohyalin (KH) granules (but no large irregular KH granules as in NEK) were abundant, adjacent to an electrondense stratum corneum. Also, synthesis of epidermal suprabasal keratins (K1 and 10;2D gels) was lower in ORS cultures, but clearly visible suprabasally by immunofluorescence along with other epidermal markers (involucrin, filaggrin, surface glycoprotein gp80, pemphigus vulgaris antigen). Basement membrane components (laminin, type IV collagen, bullous pemphigoid antigen) were detectable in both ORS and NEK in these assays. Thus, phenotypic expression was largely comparable, but, whereas terminal differentiation (keratinization) was progressing in NEK cultures limiting their lifespan, this seemed to be better controlled in ORS cultures and viable cell layers persisted resulting in longer survival time.     

Migration of melanoblasts into the developing murine hair follicle is accompanied by transient c-Kit expression.

Disruption of the c-Kit/stem cell factor (SCF) signaling pathway interferes with the survival, migration, and differentiation of melanocytes during generation of the hair follicle pigmentary unit. We examined c-Kit, SCF, and S100 (a marker for precursor melanocytic cells) expression, as well as melanoblast/melanocyte ultrastructure, in perinatal C57BL/6 mouse skin. Before the onset of hair bulb melanogenesis (i.e., stages 0-4 of hair follicle morphogenesis), strong c-Kit immunoreactivity (IR) was seen in selected non-melanogenic cells in the developing hair placode and hair plug. Many of these cells were S100-IR and were ultrastructurally identified as melanoblasts with migratory appearance. During the subsequent stages (5 and 6), increasingly dendritic c-Kit-IR cells successively invaded the hair bulb, while S100-IR gradually disappeared from these cells. Towards the completion of hair follicle morphogenesis (stages 7 and 8), several distinct follicular melanocytic cell populations could be defined and consisted broadly of (a) undifferentiated, non-pigmented c-Kit-negative melanoblasts in the outer root sheath and bulge and (b) highly differentiated melanocytes adjacent to the hair follicle dermal papilla above Auber's line. Widespread epithelial SCF-IR was seen throughout hair follicle morphogenesis. These findings suggest that melanoblasts express c-Kit as a prerequisite for migration into the SCF-supplying hair follicle epithelium. In addition, differentiated c-Kit-IR melanocytes target the bulb, while non-c-Kit-IR melanoblasts invade the outer root sheath and bulge in fully developed hair follicles.     

Establishment and characterization of outer root sheath (ORS) cell line from Jining grey goat

A new line of outer root sheath (ORS) cells was established from hair follicles of Jining grey goat by using a mechanical separation combined with enzyme digestion. Cell morphology is described at different phases. The chromosome analysis of ORS cells, identification of the ORS cells and morphological reversion test were detected at the 4th and 40th passages. The ORS cells were healthy and the growth characteristics were stable with a population doubling time of 52 h. Chromosome analysis showed that >58% of cells were diploid. Test for ORS cell line CK19 expression was positive. This newly established ORS cell line not only lays the foundation for further studying on the growth, regeneration, development law of goat hair follicle but also provides a mirror for the research of human hair in medical field.