Morphological changes in the skin and wool fibres of Merino sheep infused with mouse epidermal growth factor

Intravenous infusion of 4.5-4.7 mg of mouse epidermal growth factor (mEGF) into nine castrated male Merino sheep for 26 h resulted in complete casting of the fleeces 6-8 days later. The morphological changes which occurred in the skin were studied in skin samples taken before infusion and at intervals between 1 h and 42 days after the infusion had begun. Wool fibres from the shed fleeces were examined with the scanning electron microscope. Increased cell proliferation occurred in the epidermis and sebaceous glands, whereas the wool follicles regressed. Transient dermal haemorrhages occurred during the first 3 h of infusion. The fibre and inner root sheath in the keratogenous zone of 30-40% of the follicles were partially disrupted within the first 6 h of mEGF infusion; catagen began in all follicle bulbs within 24 h. Fibre and inner root sheath production, although markedly reduced, continued in about 60% of follicles which had partially regressed, but production ceased in the remainder in which tapered ends formed on the fibres prior to shedding. Follicles began to regenerate asynchronously 4-8 days after the beginning of infusion and completed their development during the next 3 weeks. The follicle regression and fleece casting induced by mEGF infusion, and subsequent follicle regeneration were completed more rapidly than observed previously with other depilatory agents, and, except for prolonged epidermal thickening, there was no lasting cutaneous abnormality.     

Effects of zinc deficiency on the wool growth, skin and wool follicles of pre-ruminant lambs

Two groups of 1-month-old pre-ruminant lambs of similar mean liveweights were fed identical liquid milk-replacer diets except that the zinc contents were either 5 micrograms (deficient diet) or 32 micrograms per gram of dry matter (control diet). These diets were fed for 4 weeks, after which all the lambs received the control diet for 2 weeks. In the lambs fed the deficient diet plasma zinc concentration decreased markedly during the first 2 weeks and skin lesions developed around their mouths. Autophagic vacuoles also developed in most follicle bulbs along with a variety of defects in the wool fibres and progressive inhibition of wool growth. Food intake and liveweight increase were not significantly depressed until the third and fourth weeks of feeding the deficient diet. During this period the wool was shed from the zinc-deficient lambs as a result of the fibres being degraded and distorted within thickened outer root sheaths in the distal (upper) parts of the follicles. In addition, the epidermis of the wool-bearing skin became slightly acanthotic and hyperkeratotic, although not parakeratotic. When the deficient lambs were fed the control diet for 2 weeks, their food intake, liveweight gain and plasma zinc concentration increased to almost those of the control lambs, but their rate of wool growth was still low and the epidermis had not returned to normal. Compared with previous studies the findings of this study suggest that pre-ruminant lambs may be more susceptible to the effects of zinc deficiency than ruminant lambs.     

Stimulation of the hypothalamic-pituitary-adrenal axis by epidermal growth factor

The effects of mouse epidermal growth factor (mEGF) on the hypothalamic-pituitary-adrenocortical axis were studied in vivo in conscious male rats and in vitro with cultured anterior pituitary cells. Both intravenous (i.v.) and intracerebroventricular (i.c.v.) injections of mEGF (5-20 ng: 8.3-33.3 pmol) produced significant, dose-related increases in plasma ACTH and corticosterone concentrations. The potency of mEGF is 1/20-1/50 of that of rat corticotropin-releasing factor (rCRF), and pretreatment with 150 micrograms alpha-helical CRF (9-41) completely abolished the effects of the two peptides. mEGF in concentrations ranging from 10 pM to 10 nM did not significantly affect ACTH release from dispersed anterior pituitary cells. It also failed to alter ACTH secretion in response to rCRF. These results indicate that mEGF stimulates the pituitary-adrenocortical axis through a CRF-dependent mechanism.     

Restoration of hair growth by surgical implantation of follicular dermal sheath.

The capacity of lower follicle dermal sheath to restore hair growth was tested by removing the lower halves of follicles, and then immediately implanting material containing dermal sheath cells from these bases, into the remaining upper epidermal follicle cavity. Over 60% of recipient follicles produced stout emergent vibrissa fibres and some operations resulted in multiple hair production from a single follicle. Histological examination revealed new dermal papillae within large bulb structures which were sited below the level of amputation--a feature that indicated that the new dermal papilla was derived from implanted material. For many follicles, the failure to produce emergent fibres could be accounted for after histological examination. These results provide clear evidence that lower follicle dermal sheath cells are capable of replacing those of the dermal papilla and it shows that they can do so in the context of the upper follicle. However, because elements of lower follicle epidermis were present in the implant material, the interactive sequence of events cannot be established. Dermal sheath cells have immense potential for papilla cell replacement: questions remain as to whether the distinction between sheath and papilla cells is one of context, or whether the transition requires specific external influences.     

小鼠皮肤及其毛囊早期发育的组织学观察

目的探讨小鼠皮肤及其毛囊的早期发育规律。方法采用常规石蜡切片和H-E染色技术,观察昆明系小鼠出生前后皮肤及其毛囊的形态发育。结果(1)孕龄16 d胎鼠的皮肤表面形成凹凸不平的深褶皱,但在生后3 d~5 d不仅皱褶的数量减少,而且凹陷变浅;(2)胎鼠孕龄16 d至19 d,其皮肤的表皮、真皮及皮肤总厚度呈现平稳增厚。但是,出生后,其表皮、真皮和皮肤总厚度急剧降低;在生后第1天至第9天,表皮呈现平稳增厚,而真皮则在生后快速厚度,第7天达到最高值(1861.50μm);(3)孕龄16 d的胎鼠皮肤中可观察到初级毛囊,至生后第7天其密度呈现平稳增长;与其相比,次级毛囊从18 d胎鼠开始出现,其密度增长非常迅速,出生后第7天达到1257.14/mm;毛囊的总密度与次级毛囊呈现相似的变化趋势。出生第7天后,由于毛囊的数量急剧增加,无法观察初级毛囊和次级毛囊的变化规律;(4)初级毛囊和次级毛囊的长度与深度变化在出生前后的相对缓慢,与其相比在第3天以后至第7天呈现迅速变化趋势。结论小鼠皮肤及其毛囊的生长性发育发生在胎儿晚期和生后的早期,而其周期性变化可能从出生后的第9天以后开始出现;在孕期16 d至生后第7天可能是检测毛囊特异性基因表达的最佳期。     

Migration and keratinization of cells in wool follicles

Migration of cells in wool follicles of an adult Merino sheep was studied autoradiographically in skin samples taken at intervals after an intravenous injection of [3H]thymidine. Fibre and inner root sheath cells incorporated [3H]thymidine in a cone-shape region of the follicle bulb. Labelled inner sheath cells migrated out of the bulb ahead of contemporaneous cells in the fibre and remained in advance, although to a progressively lesser extent, until the inner sheath cells sloughed into the follicle lumen. Outer root sheath cells incorporated [3H]thymidine along the length of the follicle. Cells in the proximal half of the outer sheath migrated inwards and distally and sloughed into the follicle lumen before contemporaneous inner sheath cells. Other cells in the distal half of the outer sheath migrated past the level where cells from the proximal population were shed and also sloughed into the lumen. In the most distal part of the outer sheath, which formed the epidermis-like lining of the follicle canal, little migration of cells was observed during 8 days of observation. The specific activity of tritium in fibres plucked from the same sheep at intervals after the intravenous injection of [3H]thymidine was determined by scintillation counting and assessed in terms of cell migration and hardening of the fibres. The time which the specific activity of solvent-degreased fibres reached a maximum was found to give an estimate of the time for cells in the fibre to migrate to the upper limit of the keratogenous zone. When the plucked fibres were extracted with 8 M urea the times of the maximum specific activities of the urea-dispersible and urea-insoluble material provided respectively estimates of the times at which hardening of the fibres began and ended. The effects of different planes of nutrition were examined in two other Merino sheep by radioassay of fibres plucked after intravenous injections of [3H]thymidine given after equilibration period of at least 2 months on each level of feeding. A high plane of nutrition the rate of cell migration and hastened the onset of hardening of the fibres, but prolonged the hardening process. The prolongation of the hardening process was confirmed by the specific activities of fibres plucked after intravenous injections of [35S]cystine.     

Demonstration of a considerable amount of mouse epidermal growth factor in aqueous humor

In a previous study we reported the presence of a considerable amount of mouse epidermal growth factor (mEGF) in abdominal effusion. Using our EIA system for mEGF, we identified a high level of EGF-like immunoreactive material(s) in mouse aqueous humor. This material(s) and mEGF from mouse submaxillary gland were virtually equivalent with respect to molecular weight and antigenicity. Also, on chromatofocusing analysis, the mEGF-like material(s) gave a major peak at pH 4.7 with a minor one at pH 4.2. These results demonstrate that the mEGF-like immunoreactive material(s) found in aqueous humor is a molecule identical to submaxillary gland EGF. Also, no clear difference was observed in the mEGF levels in aqueous humor between male and female. Further, sialoadenectomy did not change dramatically the EGF level in aqueous humor. From these results, it seems that mEGF found in aqueous humor may be synthesized by cells in the eyeball itself or be transported there from some site other than the submaxillary gland.     

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.     

Development of interfollicular epidermis on the surface of collagen framework of the dermis in experimental animals]

The fate of interfollicular epidermis keratinocytes which filled the hair follicles bursas of collagen dermis framework was investigated in the autotransplantation experiment. Collagen dermis framework was prepared from the skin flap. Interfollicular epidermis was detached with the help of suction method and transferred to the collagen dermis framework surface which was placed previously on the full thickness skin defect surface. It was established that in this environment keratinocytes not only developed, but some of them migrated into the hair follicle bursas cavities. It resulted in the follicular-like structures formation, cell elements of which differentiated in the manner characteristic of interfollicular epidermis. However, in spite of the fact that the epidermal cells partially retained their proliferating ability, ingrowing would completely disappear by the 15th to 20th day after the transplantation.     

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.     

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。     

Different Populations of Melanocytes Are Present in Hair Follicles and Epidermis

Melanocytes in human skin reside both in the epidermis and in the matrix and outer root sheath of anagen hair follicles. Comparative study of melanocytes in these different locations has been difficult as hair follicle melanocytes could not be cultured. In this study we used a recently described method of growing hair follicle melanocytes to characterize and compare hair follicle and epidermal melanocytes in the scalp of the same individual. Three morphologically and antigenically distinct types of melanocytes were observed in primary culture. These included (1) moderately pigmented and polydendritic melanocytes derived from epidermis; (2) small, bipolar, amelanotic melanocytes; and (3) large, intensely pigmented melanocytes; the latter two were derived from hair follicles. The three sub-populations of cells all reacted with melanocyte-specific monoclonal antibody. Epidermal and amelanotic hair follicle melanocytes proliferated well in culture, whereas the intensely pigmented hair follicle melanocytes did not. Amelanotic hair follicle melanocytes differed from epidermal melanocytes in being less differentiated, and they expressed less mature melanosome antigens. In addition, hair follicle melanocytes expressed some antigens associated with alopecia areata, but not antigens associated with vitiligo, whereas the reverse was true for epidermal melanocytes. Thus, antigenically different populations of melanocytes are present in epidermis and hair follicle. This could account for the preferential destruction of hair follicle melanocytes in alopecia areata and of epidermal melanocytes in vitiligo.     

Reprogramming adult dermis to a neonatal state through epidermal activation of β-catenin

Hair follicle formation depends on reciprocal epidermal-dermal interactions and occurs during skin development, but not in adult life. This suggests that the properties of dermal fibroblasts change during postnatal development. To examine this, we used a PdgfraEGFP mouse line to isolate GFP-positive fibroblasts from neonatal skin, adult telogen and anagen skin and adult skin in which ectopic hair follicles had been induced by transgenic epidermal activation of β-catenin (EF skin). We also isolated epidermal cells from each mouse. The gene expression profile of EF epidermis was most similar to that of anagen epidermis, consistent with activation of β-catenin signalling. By contrast, adult dermis with ectopic hair follicles more closely resembled neonatal dermis than adult telogen or anagen dermis. In particular, genes associated with mitosis were upregulated and extracellular matrix-associated genes were downregulated in neonatal and EF fibroblasts. We confirmed that sustained epidermal β-catenin activation stimulated fibroblasts to proliferate to reach the high cell density of neonatal skin. In addition, the extracellular matrix was comprehensively remodelled, with mature collagen being replaced by collagen subtypes normally present only in developing skin. The changes in proliferation and extracellular matrix composition originated from a specific subpopulation of fibroblasts located beneath the sebaceous gland. Our results show that adult dermis is an unexpectedly plastic tissue that can be reprogrammed to acquire the molecular, cellular and structural characteristics of neonatal dermis in response to cues from the overlying epidermis.     

Sostdc1 defines the size and number of skin appendage placodes

Mammary glands and hair follicles develop as ectodermal organs sharing common features during embryonic morphogenesis. The molecular signals controlling the initiation and patterning of skin appendages involve the bone morphogenetic proteins and Wnt family members, which are commonly thought to serve as inhibitory and activating cues, respectively. Here, we have examined the role of the Bmp and Wnt pathway modulator Sostdc1 in mammary gland, and hair and vibrissa follicle development using Sostdc1-null mice. Contrary to previous speculations, loss of Sostdc1 did not affect pelage hair cycling. Instead, we found that Sostdc1 limits the number of developing vibrissae and other muzzle hair follicles, and the size of primary hair placodes. Sostdc1 controls also the size and shape of mammary buds. Furthermore, Sostdc1 is essential for suppression of hair follicle fate in the normally hairless nipple epidermis, but its loss also promotes the appearance of supernumerary nipple-like protrusions. Our data suggest that functions of Sostdc1 can be largely attributed to its ability to attenuate Wnt/β-catenin signaling.     

Effects of abomasal supplements of methionine on the wool follicles and skin of wheat-fed sheep

In wheat-fed sheep, supplemented abomasally with 1.5-6.0 g methionine per day, poor formation and improper keratinization of the wool fibres were evident 4 days after the start of the methionine supplementation. This led to kinking of the fibres. Subsequently severe distortion of the fibres, accompanied by gross thickening of the outer root sheaths, occurred in the distal (upper) halves of most follicles. Within this thickened region partial degradation of the distorted fibres occurred before emergence from the skin surface, causing a marked reduction in the tensile strength of the wool. It is postulated that kinking of the fibres stimulated the accumulation of outer root sheath cells, which led to hyperactivity of the process that normally degrades the inner root sheath, so that the poorly keratinized fibres were also partly degraded. Thickening of the epidermis and cellular infiltration of the upper dermis sometimes occurred during the infusions of methionine, whereas there were negligible effects on the sebaceous and sweat glands. Disappearance of the excess accumulation of outer root sheath cells after cessation of the methionine supplementation occurred gradually following improvement in keratinization and elimination of kinking of the fibres.     

Essential roles of BMPR-IA signaling in differentiation and growth of hair follicles and in skin tumorigenesis

Hair differentiation and growth are controlled by complex reciprocal signaling between epithelial and mesenchymal cells. To better understand the requirement and molecular mechanism of BMP signaling in hair follicle development, we performed genetic analyses of bone morphogenetic protein receptor 1A (BMPR-IA) function during hair follicle development by using a conditional knockout approach. The conditional mutation of Bmpr1a in ventral limb ectoderm and its derivatives (epidermis and hair follicles) resulted in a lack of hair outgrowth from the affected skin regions. Mutant hair follicles exhibited abnormal morphology and lacked hair formation and pigment deposition during anagen. The timing of the hair cycle and the proliferation of hair matrix cells were also affected in the mutant follicles. We demonstrate that signaling via epithelial BMPR-IA is required for differentiation of both hair shaft and inner root sheath from hair matrix precursor cells in anagen hair follicles but is dispensable for embryonic hair follicle induction. Surprisingly, aberrant de novo hair follicle morphogenesis together with hair matrix cell hyperplasia was observed in the absence of BMPR-IA signaling within the affected skin of adult mutants. They developed hair follicle tumors from 3 months of age, indicating that inactivation of epidermal BMPR-IA signaling can lead to hair tumor formation. Taken together, our data provide genetic evidence that BMPR-IA signaling plays critical and multiple roles in controlling cell fate decisions or maintenance, proliferation, and differentiation during hair morphogenesis and growth, and implicate Bmpr1a as a tumor suppressor in skin tumorigenesis.     

角蛋白15在大鼠皮肤发育中表达的研究

目的研究角蛋白15(K15)在大鼠皮肤发育中的表达状况,定位表皮干细胞.方法以不同年龄大鼠背部皮肤为标本,用组织学方法,观察出生后大鼠皮肤的形态发育变化;以K15单克隆抗体为一抗,进行免疫组织化学染色,观察K15在大鼠皮肤中的表达状况.结果(1)组织学方法显示,随着年龄的增长,大鼠背部表皮细胞层数逐渐变少;在毛囊的生长周期中,以隆突区为界,毛囊上段为恒定区,下段呈周期性变化(2)免疫组化染色显示,毛囊隆突区细胞胞浆表达K15,随年龄的增长,K15阳性细胞出现在毛母质细胞区、毛囊外根鞘和表皮基底层.结论表皮干细胞位于毛囊隆突区,与表皮的更新和毛囊的周期性变化有关.     

Effects of follicular status at treatment on follicular development and ovulation in response to FSH in Spanish merino ewes

Cyclic Spanish Merino ewes were treated on Day 13 of the estrous cycle with 12 mg, i.m., FSH-P in saline (n = 9) or propylene glycol (n = 24), currently with 100 micrograms, i.m., Cloprostenol (Day 0). From Day-6 to Day 0, the ewes were observed daily by transrectal ultrasonography, after Day 0, ultrasonography was performed every 12 h for 72 h. Sizes and locations of > or = 2 mm follicles were recorded at each observation. The ovulation rate was determined by laparoscopy on Day 7 after estrus. The number of ovulations ranged from 0 to 6 in ewes treated with FSH-P in saline and from 0 to 16 in ewes receiving FSH-P in propylene glycol (P < 0.05). In the latter group, the response was bimodally distributed; about half of the females had 1 ovulation, whereas the remainder had > 4 with a mean of 7 ovulations. The ovulation rate was associated with 2 characteristics of the largest follicle present at treatment (Day 0). First, if the largest follicle on Day 0 had not changed in diameter from Day-1 to Day 0, then 7 of 9 ewes had > 3 ovulations; if the largest follicle had either increased or decreased, only 8 of 24 ewes had > 3 ovulations (P < 0.05). Second, there was a linear trend (P < 0.07) for ovulation rate to decrease as the persistence of the largest follicle at treatment increased; no ewe in which the largest follicle on Day 0 remained present for more than 36 h ovulated more than 6 follicles. As with the ovulation rate, the numbers of large follicles on Days 1.5, 2 and 2.5 varied with the interaction of change in diameter of the largest follicle on Day 0 from Day-1 to Day 0 and with vehicle. In summary, the superovulatory response was affected by the change in diameter from Day-1 to Day 0 of the largest follicle on Day 0 and the period required for that follicle to regress after treatment with FSH-P and cloprostenol.     

Epidermal and hair follicle transglutaminases. Partial characterization of soluble enzymes in newborn mouse skin

Treatment of skins of newborn mice with the neutral protease Dispase in order to separate dermis and epidermis causes pronounced changes in the levels of transglutaminase activity in the epidermis. Two soluble transglutaminases, one anionic enzyme and one cationic enzyme, of Mr approximately 90,000 and approximately 50,000, respectively, are extracted from epidermis; and the activities of both enzymes increase as a function of the time of Dispase treatment of skin. When the anionic Mr approximately 90,000 enzyme is incubated with Dispase after its chromatographic isolation from epidermal extracts, it is converted to a lower molecular weight enzyme. Hair follicles isolated from dermis prepared by a 12-h Dispase treatment of the skin of newborn mice contain two soluble cationic transglutaminases, one of which is indistinguishable from that of epidermis and the other which is not seen in epidermis. Both of these hair follicle enzymes are of Mr approximately 50,000 and appear to exist in monomeric form. They have been partially purified. Based upon these findings, we suggest that transglutaminase processing and control occur during normal differentiation of keratinocytes in epidermis and of hair follicle epidermal cells in dermis and that production of the proper forms of the enzyme may be essential to the formation of mature cornified envelopes and hair shafts, respectively.