Effects of the Mutant Gene wellhaarig in the Chimeric Mice

The mutant genewellhaarig(we) controls the formation of the waved coat in mice, which is most pronounced in homozygotes at 10 to 21 days of postnatal development. Abnormal hair growth and structure in the we/we mutant mice results from defective cell differentiation in the inner root sheath of a hair follicle. To localize the site of the we gene action, we obtained ten chimeric mice by aggregation of the early C57BL/6-2we/we and BALB/c embryos. The chimera coat was waved, shaggy, or almost normal depending on the percentage of the mutant component. In the we/we +/+ chimeric animals of the first generation (G1) aged 21 days, both mutant and normal hair phenotypes were observed, which was especially discernible in zigzag hair. Note that none of the chimeras exhibited the alternating patterns of transversely oriented stripes or patches of either mutant or normal hair; i.e., they had a mixed parental hair phenotype. We also did not observe the animals with an intermediate phenotype, which suggests a discontinuous hair formation in chimeras according to the all or nothing principle. The data obtained indicate that the dermal papilla cells of a hair follicle are the sites for the we gene action. During the embryonic development, dermal cells are strongly mixed, which accounts for the lack of the clear-cut transverse stripes of either mutant or normal hair. The mutant genewe is probably responsible for a disrupted induction signal from the dermal papilla towards ectodermal cells of a hair follicle.     

Analysis of the effects of mutant hairless genes in chimeric mice

The autosomal recessive gene hairless (hr) is responsible for the complete hairlessness in mice homozygous for this gene. Hair shedding that begins at the age of 10 days is caused by an abnormal cycle of hair follicle development disturbed at the catagen stage. This results in enhanced programmed cell death (apoptosis) and ultimately leads to the complete hair follicle destruction and shedding of all hairs by the age of three weeks. To study the phenotypic expression of the hr gene in a chimeric organism, we have obtained 12 chimeric mice hr/hr <--> +/+ by means of aggregation of early embryos hr/hr and +/+. In chimeric mice, the hair shedding has begun two days later than in the hr/hr mice. By day 23 of postnatal development, hairless areas were present on the coat of chimeric mice or the latter were completely hairless depending on the percentage of the hr/hr mutant component. In four chimeras with high content of the mutant component (68-76%), the hair shedding process was similar to that in the hr/hr mice, though it was accomplished two days later. In three chimeras with 48-51% of the mutant component, alternating hairless and hair-covered bands were observed. These data suggest that the hr gene acts in epidermal cells of a hair follicle, because epidermal cell clones in embryonic skin migrate in the lateral-ventral direction coherently and without mixing. However, some chimeras displayed a pattern which was not so clear-cut: the band borders were illegible and hairs partly covered the hairless areas. In some chimeras, the uniform thinning of the coat was observed. Analysis of the effects of the hr mutant gene in chimeric mice differing in the ratio between mutant (hr/hr) and normal (+/+) components in tissues suggests that the hr gene acts in the epidermal cells of the hair follicle. The interactions between cells have an essential effect on the mode and degree of the hr gene expression, which leads to distortion of the "ectodermal" coat pattern in chimeras.     

The Effects of Mutant Gene hairless in Chimeric Mice

The autosomal recessive gene hairless (hr) is responsible for the complete hairlessness in mice homozygous for this gene. Hair shedding that begins at the age of 10 days is caused by an abnormal cycle of hair follicle development disturbed at the catagen stage. This results in enhanced programmed cell death (apoptosis) and ultimately leads to the complete hair follicle destruction and shedding of all hairs by the age of three weeks. To study the phenotypic expression of the hr gene in a chimeric organism, we have obtained 12 chimeric mice hr/hr +/+ by means of aggregation of early embryos hr/hr and +/+. In chimeric mice, the hair shedding has begun two days later than in the hr/hr mice. By day 23 of postnatal development, hairless areas were present on the coat of chimeric mice or the latter were completely hairless depending on the percentage of the hr/hr mutant component. In four chimeras with high content of the mutant component (68–76%), the hair shedding process was similar to that in the hr/hr mice, though it was accomplished two days later. In three chimeras with 48–51% of the mutant component, alternating hairless and hair-covered bands were observed. These data suggest that the hr gene acts in epidermal cells of a hair follicle, because epidermal cell clones in embryonic skin migrate in the lateral–ventral direction coherently and without mixing. However, some chimeras displayed a pattern which was not so clear-cut: the band borders were illegible and hairs partly covered the hairless areas. In some chimeras, the uniform thinning of the coat was observed. Analysis of the effects of the hr mutant gene in chimeric mice differing in the ratio between mutant (hr/hr) and normal (+/+) components in tissues suggests that the hrgene acts in the epidermal cells of the hair follicle. The interactions between cells have an essential effect on the mode and degree of the hr gene expression, which leads to distortion of the ectodermal coat pattern in chimeras.     

The serine protease Corin is a novel modifier of the Agouti pathway

The hair follicle is a model system for studying epithelial-mesenchymal interactions during organogenesis. Although analysis of the epithelial contribution to these interactions has progressed rapidly, the lack of tools to manipulate gene expression in the mesenchymal component, the dermal papilla, has hampered progress towards understanding the contribution of these cells. In this work, Corin was identified in a screen to detect genes specifically expressed in the dermal papilla. It is expressed in the dermal papilla of all pelage hair follicle types from the earliest stages of their formation, but is not expressed elsewhere in the skin. Mutation of the Corin gene reveals that it is not required for morphogenesis of the hair follicle. However, analysis of the ;dirty blonde' phenotype of these mice reveals that the transmembrane protease encoded by Corin plays a critical role in specifying coat color and acts downstream of agouti gene expression as a suppressor of the agouti pathway.     

The mutant gene "wal" is active in cells of mouse hair follicles

In order to determine the place of action of the mutant gene waved alopecia (wal), we have obtained chimeric wal/wal c/c Gpi-1aa<-->+/+ C/C Gpi-1bb animals by aggregation of eight-cellular embryos of BALB/c-wal/wal mice and CBA (+/+) mice. The presence or absence of the chimeric structure was determined from the mosaic nature of fur color and hair structure, as well as on the basis of the presence of electrophoretically distinct variants of glucosephosphate isomerase in blood. Chimeras had alternating transverse patches of different lengths and widths consisting of curly (genotype wal/wal) or straight (genotype +/+) hairs. The percentage of cells with wal/wal mutant genotype in chimeras established on the basis of glucosephosphate isomerase isozymes varied from 10 to 80%. A higher percentage of the parental wal/wal component in chimeras correlated with the number of patches having wavy hairs. Analysis of the fur pattern represented by the alternation of transverse patches of wavy or straight hairs in chimeric wal/wal (+/+ mice has shown that mutant gene wal acts in ectodermal cells of hair follicles.     

黄芪、何首乌、女贞子、菟丝子混合提取物对体外培养毛囊生长的影响及药理作用研究

目的:通过体内外实验探讨黄芪、何首乌、女贞子、菟丝子混合中药提取物对毛囊增殖的影响作用以及其作用机理。方法:通过体外培养的C57BL/6小鼠毛囊器官模型观察不同浓度中药提取物对毛囊生长的影响;采用MTT法测定不同浓度中药提取物对毛乳头增殖的影响;蛋白免疫印迹法(Western Blot)和ELISA检测中药提取物对毛乳头细胞分泌肝细胞生长因子(HGF)的影响。结果:中药提取物能够刺激体外培养的小鼠毛囊的生长,800μg/mL浓度的促进作用最强;160μg/mL中药提取物对毛乳头细胞的增殖作用最强,与米诺地尔、齐墩果酸阳性对照存在显著性差异(P0.05)。而且,药提取物促进了毛乳头细胞分泌HGF。结论:黄芪、何首乌、女贞子、菟丝子混合中药提取物在促进毛发生长中起到重要作用,促进毛乳头细胞增殖和分泌HGF是促进毛囊生长的可能性药理机制。     

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.     

Roles for PDGF-A and sonic hedgehog in development of mesenchymal components of the hair follicle.

Skin appendages, such as hair, develop as a result of complex reciprocal signaling between epithelial and mesenchymal cells. These interactions are not well understood at the molecular level. Platelet-derived growth factor-A (PDGF-A) is expressed in the developing epidermis and hair follicle epithelium, and its receptor PDGF-Ralpha is expressed in associated mesenchymal structures. Here we have characterized the skin and hair phenotypes of mice carrying a null mutation in the PDGF-A gene. Postnatal PDGF-A-/- mice developed thinner dermis, misshapen hair follicles, smaller dermal papillae, abnormal dermal sheaths and thinner hair, compared with wild-type siblings. BrdU labeling showed reduced cell proliferation in the dermis and in the dermal sheaths of PDGF-A-/- skin. PDGF-A-/- skin transplantation to nude mice led to abnormal hair formation, reproducing some of the features of the skin phenotype of PDGF-A-/- mice. Taken together, expression patterns and mutant phenotypes suggest that epidermal PDGF-A has a role in stimulating the proliferation of dermal mesenchymal cells that may contribute to the formation of dermal papillae, mesenchymal sheaths and dermal fibroblasts. Finally, we show that sonic hedgehog (shh)-/- mouse embryos have disrupted formation of dermal papillae. Such embryos fail to form pre-papilla aggregates of postmitotic PDGF-Ralpha-positive cells, suggesting that shh has a critical role in the assembly of the dermal papilla.     

Polyclonal origin and hair induction ability of dermal papillae in neonatal and adult mouse back skin

Hair follicle development and growth are regulated by Wnt signalling and depend on interactions between epidermal cells and a population of fibroblasts at the base of the follicle, known as the dermal papilla (DP). DP cells have a distinct gene expression signature from non-DP dermal fibroblasts. However, their origins are largely unknown. By generating chimeric mice and performing skin reconstitution assays we show that, irrespective of whether DP form during development, are induced by epidermal Wnt activation in adult skin or assemble from disaggregated cells, they are polyclonal in origin. While fibroblast proliferation is necessary for hair follicle formation in skin reconstitution assays, mitotically inhibited cells readily contribute to DP. Although new hair follicles do not usually develop in adult skin, adult dermal fibroblasts are competent to contribute to DP during hair follicle neogenesis, irrespective of whether they originate from skin in the resting or growth phase of the hair cycle or skin with β-catenin-induced ectopic follicles. We propose that during skin reconstitution fibroblasts may be induced to become DP cells by interactions with hair follicle epidermal cells, rather than being derived from a distinct subpopulation of cells.     

Interaction of mutant genes Fgf5(go-Y), we, and wal changes the duration of hair growth cycles in mice

Mutant gene wallhaarig (wa) was acting as a modifier of the mutant gene waved alopecia (wal), substantially increasing hair loss rate in mice, as was previously shown in our laboratory. The current paper is devoted to a study of mutant gene angora- Y(Fgf5(go-Y)), which had extended anagen stage of the first and second generations hair growth cycles in triple heterozygotes (Fgf5(go-Y)/Fgf5(go-Y) we/we wal/wal). First generation guard hair in triple homozygotes had their anagen stage 4 days longer than the same stage in double homozygotes (+/+ we/we wal/wal). Hair loss started at a catagen stage in double homozygotes, while it started in triple homozygotes at the end of the same stage or even in a telogen. Such mutant gene interaction in hair follicle morphogenesis led to a partial recovery of a body hair coat in triple homozygotes.     

The site of the action of the angora-Y gene and its interaction with the fuzzy-Y gene in the mouse

The site of action of the goY mutant gene was determined in the aggregation chimaeras C57BL-goY/goY----DBA (+/+). Chimerism was detected by mosaicism of coat pigmentation and electrophoretic pattern of glucose phosphate isomerase. In 28-day-old chimaeras the regions of light-brown coat alternated black coat, stripes of short hairs alternated those of long hairs. These stripes of different length and width extended from spine in lateral-ventral direction. The hairs plucked from long hairs stripes had a similar length that those of goY/goY mice of same age, but the hairs plucked from short hair stripes corresponded to the hair length of +/+ mice. These data show that the goY gene acts in epidermal cells of hair follicles and its expression is autonomous. It has been established that in double homozygotes goY/goYfzY/fzY both mutant genes are expressed: the considerable increase of hair length as compared to norm--the effect of the goY gene and curly coat--the effect of the fzY gene. In goY/goYfzY/fzY mice during the formation of G1 guard hairs the incomplete expression of the goY gene is observed that is due to the suppression of hair growth by the fzY mutant gene. The fzY gene does not suppress the growth of G2 hairs and therefore the full expression of the goY gene occurs in goY/goYfzY/fzY adult mice.     

Sostdc1 Secreted from Cutaneous Lymphatic Vessels Acts as a Paracrine Factor for Hair Follicle Growth

In our previous study, we found that lymphatic vessels stimulate hair follicle growth through paracrine effects on dermal papilla cells. However, the paracrine factors secreted from cutaneous lymphatic vessels that can activate dermal papilla cells are still unknown. In this study, we investigated whether lymphatic endothelial cells might secrete paracrine factors that activate dermal papilla cells in vitro. We found that Sostdc1 was more expressed in lymphatic endothelial cells compared with blood vascular endothelial cells. In addition, Sostdc1 expression levels were significantly increased during the anagen phase in the back skin of C57BL/6J mice, as compared to the telogen phase. We also observed that incubation of dermal papilla cells with 200 ng/mL Sostdc1 for 72 h induced the expression levels of Lef-1, a downstream target of Wnt signaling. Taken together, our results reveal that Sostdc1, a BMP antagonist, secreted from cutaneous lymphatic vessels, may act as a paracrine factor for hair follicle growth.     

The we gene is a modifier of the wal gene in mice

Interaction of gene wellhaarig (we) with genes waved alopecia (wal) and hairless (hr) was studied in mice. The mutant gene we is responsible for the development of a specific waved coat in homozygotes. Homozygous mice carrying mutant gene wal also have a wavy coat, though a partial alopecia develops with time in these animals. In homozygotes for the hr gene, hair loss is observed beginning from the age of ten days. A series of crosses we/we and wal/wal yielded animals with we/+wal/wal and we/we wal/wal genotypes. In mice we/+wal/wal carrying gene we at a single dose, alopecia is accelerated significantly as compared to the single-dose homozygotes +/+wal/wal. In we/we wal/wal mice, alopecia starts earlier than in we/+wal/wal mice; by the age of one month, the double homozygotes are almost hairless except for small body areas covered with a sparse coat. In addition, curliness of the first-generation hair in mice we/we wal/wal is much more expressed than in +/+wal/wal and we/we+/+ mice. The obtained evidence suggests that the we gene is a modifier of the wal gene because the former enhances the effects of the wal gene, which is confirmed by the earlier onset of alopecia and progression of the latter in mice having the we/+wal/wal genotype and especially in we/we wal/wal animals. The we/we hr/+ mice do not differ in coat from we/we+/+ mice; in both cases, the coat is wavy. The coat of double homozygotes we/we hr/hr, is similar to that of we/we+/+ mice until ten days of age, when the signs of alopecia appear. By the age of 21 days, mice we/we hr/hr have lost their coat completely like mice +/+ hr/hr. Hence, the we gene is a modifier of the wal gene though it does not interact with hr gene during the coat formation.     

Generation of pigmented stripes in albino mice by retroviral marking of neural crest melanoblasts.

The pigment cells of the skin are derived from melanoblasts which originate in the neural crest. The dorsoventral migration of melanoblasts has been visualized in pigment stripes seen in aggregation chimeras, and the width of these bands has suggested that the entire pigmentation of the coat is derived from a small number of founder cells. We have generated mosaic mice by marking single melanoblasts in utero to gain information on the clonal history of pigment-forming cells. A retroviral vector carrying the human tyrosinase gene was constructed and microinjected into neurulating albino mouse embryos. Albino mice are devoid of pigmentation due to deficiency of tyrosinase. Thus, transduction of the wild-type gene into the otherwise normal melanoblasts should rescue the mutant phenotype, giving rise to patches of pigmentation, which correspond to the area colonized by the mitotic progeny of a marked clone. Mosaic animals derived from the injected embryos indeed showed pigmented bands with a width strikingly similar to the 'standard' stripes seen in aggregation chimeras. These results are consistent with the notion that the unit width bands seen in aggregation chimeras represent the clonal progeny of a single melanoblast and verify Mintz's (1967) conclusion that a few founder melanoblasts give rise to coat pigmentation. The pigment cells of the eye are of dual origin: the melanocytes in choroid and outer layer of the iris are derived from the neural crest and those in the pigment layer of the retina from the neuroepithelium of the optic cup. Marked clones in both lineages were observed in the eyes of many mosaic animals.     

Cultured dermal papilla cells induce follicle formation and hair growth by transdifferentiation of an adult epidermis.

Adult rat pelage follicle dermal papilla cells induced follicle neogenesis and external hair growth when associated with adult footpad skin epidermis. They thus demonstrated a capacity to completely change the structural arrangement and gene expression of adult epidermis--an ability previously undocumented for cultured adult cells. Isolation chambers ensured that de novo follicle formation must have occurred by eliminating the possibility of cellular contributions, and/or inductive influences, from local skin follicles. These findings argue against previous suggestions of vibrissa follicle specificity, and imply that the potential for hair follicle induction may be common to all adult papilla cells.     

Characterization of Hair Follicle Development in Engineered Skin Substitutes

Generation of skin appendages in engineered skin substitutes has been limited by lack of trichogenic potency in cultured postnatal cells. To investigate the feasibility and the limitation of hair regeneration, engineered skin substitutes were prepared with chimeric populations of cultured human keratinocytes from neonatal foreskins and cultured murine dermal papilla cells from adult GFP transgenic mice and grafted orthotopically to full-thickness wounds on athymic mice. Non-cultured dissociated neonatal murine-only skin cells, or cultured human-only skin keratinocytes and fibroblasts without dermal papilla cells served as positive and negative controls respectively. In this study, neonatal murine-only skin substitutes formed external hairs and sebaceous glands, chimeric skin substitutes formed pigmented hairs without sebaceous glands, and human-only skin substitutes formed no follicles or glands. Although chimeric hair cannot erupt readily, removal of upper skin layer exposed keratinized hair shafts at the skin surface. Development of incomplete pilosebaceous units in chimeric hair corresponded with upregulation of hair-related genes, LEF1 and WNT10B, and downregulation of a marker of sebaceous glands, Steroyl-CoA desaturase. Transepidermal water loss was normal in all conditions. This study demonstrated that while sebaceous glands may be involved in hair eruption, they are not required for hair development in engineered skin substitutes.     

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model ^{5, 6, 11}, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.     

Capability of cultivated human hair-follicle cells to integrate with skin structure in vivo

In the present work, we labeled human epidermal keratinocytes and dermal papilla cells in order to study their behavior after intradermal transplantation. The cells were transduced by lentiviral vectors that bore a marker gene that encodes green fluorescent protein (copGFP) or red fluorescent protein (DsRed). A portion of the transgene expressing cells was evaluated by flow cytometry. The proposed genetic constructions have allowed one to achieve high efficiency (>95%) of the transduction of hair follicle cells. The in vitro transduced cells were injected under epidermis of human skin fragments, after which these fragments were transplanted under the skin of immunodeficient mice. The injected epidermal keratinocytes were found mainly in hair follicles and partially in the zone of interfollicular epidermis, while dermal papilla cells were found in the papilla of the derma. The results of the present study have shown that the chosen genetic constructions obtained based on human immunodeficiency lentivirus are capable of the effective and stable transduction of human skin cells. The injected cells survived and were found in the corresponding skin structures.     

Role of the vitamin D receptor in hair follicle biology

The vitamin D receptor (VDR) is expressed in numerous cells and tissues, including the skin. The critical requirement for cutaneous expression of the VDR has been proven by investigations in mice and humans lacking functional receptors. These studies demonstrate that absence of the VDR leads to the development of alopecia. The hair follicle is formed by reciprocal interactions between an epidermal placode, which gives rise to the hair follicle keratinocytes and the underlying mesoderm which gives rise to the dermal papilla. Hair follicle morphogenesis ends the second week of life in mice. Studies in VDR null mice have failed to demonstrate a cutaneous abnormality during this period of hair follicle morphogenesis. However, VDR null mice are unable to initiate a new hair cycle after the period of morphogenesis is complete, therefore, do not grow new hair. Investigations in transgenic mice have demonstrated that restricted expression of the VDR to keratinocytes is capable of preventing alopecia in the VDR null mice, thus demonstrating that the epidermal component of the hair follicle requires VDR expression to maintain normal hair follicle homeostasis. Studies were then performed to determine which regions of the VDR were required for these actions. Investigations in mice lacking the first zinc finger of the VDR have demonstrated that they express a truncated receptor containing an intact ligand binding and AF2 domain. These mice are a phenocopy of mice lacking the VDR, thus demonstrate the critical requirement of the DNA binding domain for hair follicle homeostasis. Transgenic mice expressing VDRs with mutations in either the ligand-binding domain or the AF2 domain were generated. These investigations demonstrated that mutant VDRs incapable of ligand-dependent transactivation were able to prevent alopecia. Investigations are currently underway to define the mechanism by which the unliganded VDR maintains hair follicle homeostasis.