EDAR参与绵羊毛发性状与生长的调节

绵羊的背部、耳部和腹股沟部的毛发性状、生长速度存在差异,背部毛发弯曲、细长、密度高、生长速度快,耳部、腹股沟部毛发粗直、密度低、生长速度慢。研究表明,EDA/EDAR、IGFBP5/Krox 20、WNT等介导的信号通路及毛发角蛋白基因对毛发纤维弯曲的形成有重要的影响。本研究利用实时荧光定量PCR、原位杂交技术、Western 印迹和免疫组织化学等技术,对外异蛋白受体（ectodysplasin A receptor,EDAR）在绵羊背部、耳部和腹股沟皮肤中的mRNA、蛋白质表达水平和定位进行研究,以探讨EDAR与绵羊毛发的生长和性状的关系。实时荧光定量PCR结果显示,EDAR在绵羊背部皮肤中相对基因表达量是绵羊腹股沟皮肤的4.9倍(P＜ 0.01),耳部是腹股沟部的1.4倍（P＜0.05）,背部是耳部的3.4倍（P＜0.05）;原位杂交和免疫组化结果表明,EDAR基因mRNA和蛋白质在背部、耳部和腹股沟部毛囊均有表达。根据光密度值可知,背部表达量最高,耳部次之,而腹股沟部最低;Western 印迹结果显示,绵羊皮肤组织蛋白质提取物中存在与兔抗EDAR多克隆抗体发生免疫阳性反应的蛋白条带,绵羊皮肤背部平均蛋白质表达量最高,耳部次之,而腹股沟部最低,差异极显著(P ＜ 0.01)。研究结果提示,EDAR可能参与绵羊毛发卷曲的形成和调控,对毛发密度、生长速度等可能也有影响。     

The role of Wnt/β‐catenin signaling in proliferation and regeneration of the developing basilar papilla and lateral line

Canonical Wnt/β‐catenin signaling has been implicated in multiple developmental events including the regulation of proliferation, cell fate, and differentiation. In the inner ear, Wnt/β‐catenin signaling is required from the earliest stages of otic placode specification through the formation of the mature cochlea. Within the avian inner ear, the basilar papilla (BP), many Wnt pathway components are expressed throughout development. Here, using reporter constructs for Wnt/β‐catenin signaling, we show that this pathway is active throughout the BP (E6‐E14) in both hair cells (HCs) and supporting cells. To characterize the role of Wnt/β‐catenin activity in developing HCs, we performed gain‐ and loss‐of‐function experiments in vitro and in vivo in the chick BP and zebrafish lateral line systems, respectively. Pharmacological inhibition of Wnt signaling in the BP and lateral line neuromasts during the periods of proliferation and HC differentiation resulted in reduced proliferation and decreased HC formation. Conversely, pharmacological activation of this pathway significantly increased the number of HCs in the lateral line and BP. Results demonstrated that this increase was the result of up‐regulated cell proliferation within the Sox2‐positive cells of the prosensory domains. Furthermore, Wnt/β‐catenin activation resulted in enhanced HC regeneration in the zebrafish lateral line following aminoglycoside‐induced HC loss. Combined, our data suggest that Wnt/β‐catenin signaling specifies the number of cells within the prosensory domain and subsequently the number of HCs. This ability to induce proliferation suggests that the modulation of Wnt/β‐catenin signaling could play an important role in therapeutic HC regeneration. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 438–456, 2014     

Establishment of an in vitro organoid model of dermal papilla of human hair follicle

Human hair dermal papilla (DP) cells are specialized mesenchymal cells that play a pivotal role in hair regeneration and hair cycle activation. The current study aimed to first develop three‐dimensional (3D) DP spheroids (DPS) with or without a silk–gelatin (SG) microenvironment, which showed enhanced DP‐specific gene expression, resulting in enhanced extracellular matrix (ECM) production compared with a monolayer culture. We tested the feasibility of using this DPS model for drug screening by using minoxidil, which is a standard drug for androgenic alopecia. Minoxidil‐treated DPS showed enhanced expression of growth factors and ECM proteins. Further, an attempt has been made to establish an in vitro 3D organoid model consisting of DPS encapsulated by SG hydrogel and hair follicle (HF) keratinocytes and stem cells. This HF organoid model showed the importance of structural features, cell–cell interaction, and hypoxia akin to in vivo HF. The study helped to elucidate the molecular mechanisms to stimulate cell proliferation, cell viability, and elevated expression of HF markers as well as epithelial–mesenchymal crosstalks, demonstrating high relevance to human HF biology. This simple in vitro DP organoid model system has the potential to provide significant insights into the underlying mechanisms of HF morphogenesis, distinct molecular signals relevant to different stages of the hair cycle, and hence can be used for controlled evaluation of the efficacy of new drug molecules.     

A second KRT71 allele in curly coated dogs

Major characteristics of coat variation in dogs can be explained by variants in only a few genes. Until now, only one missense variant in the KRT71 gene, p.Arg151Trp, has been reported to cause curly hair in dogs. However, this variant does not explain the curly coat in all breeds as the mutant ¹⁵¹Trp allele, for example, is absent in Curly Coated Retrievers. We sequenced the genome of a Curly Coated Retriever at 22× coverage and searched for variants in the KRT71 gene. Only one protein‐changing variant was present in a homozygous state in the Curly Coated Retriever and absent or present in a heterozygous state in 221 control dogs from different dog breeds. This variant, NM_001197029.1:c.1266_1273delinsACA, was an indel variant in exon 7 that caused a frameshift and an altered and probably extended C‐terminus of the KRT71 protein NP_001183958.1:p.(Ser422ArgfsTer?). Using Sanger sequencing, we found that the variant was fixed in a cohort of 125 Curly Coated Retrievers and segregating in five of 14 additionally tested breeds with a curly or wavy coat. KRT71 variants cause curly hair in humans, mice, rats, cats and dogs. Specific KRT71 variants were further shown to cause alopecia. Based on this knowledge from other species and the predicted molecular consequence of the newly identified canine KRT71 variant, it is a compelling candidate causing a second curly hair allele in dogs. It might cause a slightly different coat phenotype than the previously published p.Arg151Trp variant and could potentially be associated with follicular dysplasia in dogs.     

Appearance of hair follicle-inducible mesenchymal cells in the rat embryo

Rat vibrissa follicle morphogenesis starts around 13 days of gestation. By day 14 mesenchymal cells have already aggregated as 'condensations' beneath the initial hair bud. Some of the mesenchymal cells will form a dermal papilla, having profound effects on hair follicle formation. The appearance of follicle-inducing mesenchymal cells in the process of vibrissa follicle development was examined. Mesenchymal cells were isolated from the developing site of vibrissa follicles at 13 days or at later stages and amplified in mass culture, harvested and transplanted in association with the epithelium. It was demonstrated that 13-day mesenchymal cells did not induce any hair bulbs but those from 14 days or later stages could induce hair-producing new bulbs or new follicles depending on the association with the follicle epithelium or with the glabrous sole epidermis of the adult rats, respectively. Further, clones having hair bulb-inducing ability were obtained from 14- and 15-day mass-cultured mesenchymal cells. Based on these and other results, it was concluded that mesenchymal cells having follicle-inducing ability are present at least by 14 days in the future whisker pad region. This suggests that the differentiation of the dermal papilla cells must start before the initial hair bud stage.     

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.     

Activation of Wnt/β‐catenin signalling is required for TGF‐β/Smad2/3 signalling during myofibroblast proliferation

Fibrosis in animal models and human diseases is associated with aberrant activation of the Wnt/β‐catenin pathway. Despite extensive research efforts, effective therapies are still not available. Myofibroblasts are major effectors, responsible for extracellular matrix deposition. Inhibiting the proliferation of the myofibroblast is crucial for treatment of fibrosis. Proliferation of myofibroblasts can have many triggering effects that result in fibrosis. In recent years, the Wnt pathway has been studied as an underlying factor as a primary contributor to fibrotic diseases. These efforts notwithstanding, the specific mechanisms by which Wnt‐mediated promotes fibrosis reaction remain obscure. The central role of the transforming growth factor‐β (TGF‐β) and myofibroblast activity in the pathogenesis of fibrosis has become generally accepted. The details of interaction between these two processes are not obvious. The present investigation was conducted to evaluate the level of sustained expression of fibrosis iconic proteins (vimentin, α‐SMA and collagen I) and the TGF‐β signalling pathway that include smad2/3 and its phosphorylated form p‐smad2/3. Detailed analysis of the possible molecular mechanisms mediated by β‐catenin revealed epithelial–mesenchymal transition and additionally demonstrated transitions of fibroblasts to myofibroblast cell forms, along with increased activity of β‐catenin in regulation of the signalling network, which acts to counteract autocrine TGF‐β/smad2/3 signalling. A major outcome of this study is improved insight into the mechanisms by which epithelial and mesenchymal cells activated by TGFβ1‐smad2/3 signalling through Wnt/β‐catenin contribute to lung fibrosis.     

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.     

Role of Sonic hedgehog signaling in epithelial and mesenchymal development of hair follicles in an organ culture of embryonic mouse skin

Studies with gene knockout mice have shown that Sonic hedgehog (Shh) is required for early development of hair follicles, but the role of this gene in the late stages of follicle development is not clear. By using an organ culture system of embryonic mouse skin, the role of Shh signaling in the early and late stages of follicle development was investigated. In the early stage of follicle development, the downward growth of the follicular epithelium was suppressed by cyclopamine, an inhibitor of Shh signaling, and accelerated by recombinant Shh. In addition, cyclopamine impaired dermal papilla formation, accompanied by the rearrangement of papilla cells, but not the elongation of the follicular epithelium at the later stage. These results suggest that Shh signaling is required for the proliferation of epithelial cells in the early development of hair follicles and for the morphogenetic movement of mesenchymal cells at the later stage of follicle development.     

Defining the identity of mouse embryonic dermal fibroblasts

Embryonic dermal fibroblasts in the skin have the exceptional ability to initiate hair follicle morphogenesis and contribute to scarless wound healing. Activation of the Wnt signaling pathway is critical for dermal fibroblast fate selection and hair follicle induction. In humans, mutations in Wnt pathway components and target genes lead to congenital focal dermal hypoplasias with diminished hair. The gene expression signature of embryonic dermal fibroblasts during differentiation and its dependence on Wnt signaling is unknown. Here we applied Shannon entropy analysis to identify the gene expression signature of mouse embryonic dermal fibroblasts. We used available human DNase‐seq and histone modification ChiP‐seq data on various cell‐types to demonstrate that genes in the fibroblast cell identity signature can be epigenetically repressed in other cell‐types. We found a subset of the signature genes whose expression is dependent on Wnt/β‐catenin activity in vivo. With our approach, we have defined and validated a statistically derived gene expression signature that may mediate dermal fibroblast identity and function in development and disease. genesis 54:415–430, 2016. © 2016 Wiley Periodicals, Inc.     

Cultured human and rat tooth papilla cells induce hair follicle regeneration and fiber growth

The mesenchymal-epithelial interactions that characterize the early stages of tooth and hair follicle morphogenesis share certain similarities, and there is increasing evidence that mesenchymal cells derived from both mature structures retain interactive and stem cell-like properties. This study aimed to gauge the cross-appendage inductive capabilities of cultured tooth dental papilla (or pulp) cells from different species and ages of donor. Adult human and juvenile rat tooth papilla cells were implanted into surgically inactivated hair follicles within two different microenvironments. The human cells interacted with follicle epithelium to regenerate new end bulbs and create multiple differentiated hair fibers. Rodent tooth dental cells also induced new epithelial matrix structures and stimulated de novo hair formation. However, in many instances they also elicited mineralization and bone formation, a phenomenon that appeared to relate to their donor's age; the type of tooth of origin; and the host environment. Taken together, this study reveals that cultured dental papilla cells from postnatal mammals (adult, juvenile, and newborn) retain inductive molecular signals that must be common to both hair and teeth follicles. It highlights the stem cell-like qualities and morphogenetic abilities of tooth and hair follicle cells from mature humans, and their capacity for cross-appendage and interspecies communication and interaction. Besides the developmental implications, the present findings have relevance for stem cell biology, hair growth, tissue repair, and other biotechnologies. Moreover, the critical importance of considering the local microenvironment in which different cells/tissues are naturally or experimentally engineered is firmly demonstrated.     

Platelet sonicates activate hair follicle stem cells and mediate enhanced hair follicle regeneration

An increasing number of studies show that platelet‐rich plasma (PRP) is effective for androgenic alopecia (AGA). However, the underlying cellular and molecular mechanisms along with its effect on hair follicle stem cells are poorly understood. In this study, we designed to induce platelets in PRP to release factors by calcium chloride (PC) or by sonication where platelet lysates (PS) or the supernatants of platelet lysate (PSS) were used to evaluate their effect on the hair follicle activation and regeneration. We found that PSS and PS exhibited a superior effect in activating telogen hair follicles than PC. In addition, PSS injection into the skin activated quiescent hair follicles and induced K15⁺ hair follicle stem cell proliferation in K14‐H2B‐GFP mice. Moreover, PSS promoted skin‐derived precursor (SKP) survival in vitro and enhanced hair follicle formation in vivo. In consistence, protein array analysis of different PRP preparations revealed that PSS contained higher levels of 16 growth factors (out of 41 factors analysed) than PC, many of them have been known to promote hair follicle regeneration. Thus, our data indicate that sonicated PRP promotes hair follicle stem cell activation and de novo hair follicle regeneration.     

褪黑素对绒山羊皮肤FGF5等绒毛生长相关基因的影响

目的:旨在分析埋植褪黑素后对绒山羊皮肤绒毛生长相关基因表达的影响,进而探讨褪黑素促绒毛生长的机理。方法:以内蒙古白绒山羊为研究对象,选取体况相近的4月龄母羔羊10只,按试验要求随机分为2组,处理组按照2mg/kg BW(体重)的剂量每两个月耳后皮下埋植褪黑素,对照组不埋植,试验时间为1年。实验开始后每月采集绒山羊体侧皮肤和毛样,利用荧光定量PCR技术检测9个绒毛生长相关基因的表达变化。结果:(1)埋植褪黑素后各个基因的平均表达水平都有所上调,其中显著上调FGF5(P<0.0001),而对β-catenin、MSX-2、Wnt10b、BMP2、BMPRIB、NTRK3、Delta1、hairless等无显著影响(P>0.05);(2)埋植褪黑素明显提高了FGF5与hairless的相关性,并且使FGF5与β-catenin、NTRK3的相关性由负相关变成正相关,与Wnt10b的相关性降低,FGF5与其余4个基因的相关性变化不大。结论:结果提示,褪黑素上调绒山羊皮肤FGF的表达,影响FGF5与hairless、β-catenin、NTRK3之间的相关性;山羊中FGF5基因对被毛表型的作用与其他动物不同;褪黑素对复杂组织和单个细胞的作用不同;MAPK信号通路是受褪黑素影响最大的一个信号途径。推测褪黑素可能通过调节绒山羊皮肤中RORα来发挥作用,影响FGF5与hairless、β-catenin、NTRK3和Wnt10b等的相关性,同时抑制MSX2和BMP2的功能,进而促进绒毛的生长。     

Ectopic expression of ROR1 prevents cochlear hair cell loss in guinea pigs with noise‐induced hearing loss

Noise‐induced hearing loss (NIHL) is one of the most frequent disabilities in industrialized countries. Evidence shows that hair cell loss in the auditory end organ is responsible for the majority of various ear pathological conditions. The functional roles of the receptor tyrosine kinase ROR1 have been underscored in various tumours. In this study, we evaluated the ability of ROR1 to influence cochlear hair cell loss of guinea pigs with NIHL. The NIHL model was developed in guinea pigs, with subsequent measurement of the auditory brainstem response (ABR). Gain‐of‐function experiments were employed to explore the role of ROR1 in NIHL. The interaction between ROR1 and Wnt5a and their functions in the cochlear hair cell loss were further analysed in response to alteration of ROR1 and Wnt5a. Guinea pigs with NIHL demonstrated elevated ABR threshold and down‐regulated ROR1, Wnt5a and NF‐κB p65. The up‐regulation of ROR1 was shown to decrease the cochlear hair cell loss and the expression of pro‐apoptotic gene (Bax, p53) in guinea pig cochlea, but promoted the expression of anti‐apoptotic gene (Bcl‐2) and the fluorescence intensity of cleaved‐caspase‐3. ROR1 interacted with Wnt5a to activate the NF‐κB signalling pathway through inducing phosphorylation and translocation of p65. Furthermore, Wnt5a overexpression decreased the cochlear hair cell loss. Collectively, this study suggested the protection of overexpression of ROR1 against cochlear hair cell loss in guinea pigs with NIHL via the Wnt5a‐dependent NF‐κB signalling pathway.