| BackgroundThe hair follicle is one of the most complex miniorgans of the human body.The most obvious function of hair follicle is to produce a hair shaft,which exerts a wide range of functions including physical protection, thermal insulation, camouflage, dispersion of sweat and sebum, sensory and tactile functions, and social interactions. Among them, the most important function of the shaft acts as a physical medium of social communication. Hair loss, as well as unwanted hair growth (hirsutism, hypertrichosis), has been a widespread problem. According to one calculation, androgenetic alopecia on its own eventually affects approximately 50% of the world's adult population. Although hair disorders are not life threatening, their profound impact on social interactions and patients psychological well being is undeniable. However, until very recently, there is no very effective therapy to get a satisfactory treatment, which may be due to our poor understanding of the molecules and pathways regulating hair follicle formation and hair growth. Consequently, It is extremely urgent to deepen our knowledge of the molecular principles of hair follicle morphogenesis and renewal, which will hopefully lead to the development of new therapeutic approaches or more effective "hair drugs" for the treatment of hair disorders.Increased evidence suggests that hair follicle morphogenesis and the initiation of a new growth phase in the postnatal hair follicles show many similar features. Many growth factors and their receptors that are involved in signaling exchanges between the epithelium and the mesenchyme during hair follicle development also regulate the cyclic activity of postnatal hair follicles. So the molecular principles of hair follicle induction and morphogenesis are also suitable for hair renewal.The formation of hair follicles occurs during embryogenesis and relies on a series of signals that cause fate changes between dermal cells and overlying surface epithelial cells, ultimately resulting in differentiation of the hair shaft, root sheaths, and dermal papilla. These signals include secreted molecules of the Wnt/wingless family,the hedgehog family,and members of the TGF-β/BMP, FGF and TNF families(Eda/Edar), among which the most important signal is Wnt/wingless family. Multiple knockout and transgenic mouse models have shown by now that the Wnt signaling operates as the key stimulator of the hair follicle development.Wnts are a large family of secreted signaling molecules with homology to the fly Wingless protein, which are transduced through at least three distinct intracellular signaling pathways including the canonical Wnt/β-catenin pathway, the Wnt/Ca2+ pathway, and the Wnt/polarity pathway.The canonical Wnt/β-catenin pathway depends on the levels ofβ-catenin within the cell. In the absence of Wnt,β-catenin is targeted for degradation by a multi-protein destruction complex.While in the presence of Wnt, the Wnt signaling antagonizes the destruction complex, leading to the accumulation ofβ-catenin in the cytoplasm,which enables it to translocate to the nucleus and activate target genes. The canonical Wnt/β-catenin signalling provides the master switch for hair follicle fate, as ectopic epithelial expression of the secreted Wnt inhibitor Dkkl or lack of epidermalβ-catenin expression results in the absence of hair follicle induction, Conversely, the forced expression of a stabilized form ofβ-catenin causes the strongly enhanced placode formation, because the epidermal keratinocytes globally adopt a hair follicle fate. However, up to this date, there are 19 known family members in humans and mice, and it is still not known exactly which member of the Wnt family is responsible for the hair follicle initiation and whether it arises intradermally or intraepidermally.It has been carried out a comprehensive survey of all currently identified mouse Wnt genes expression in embryonic and postnatal skin and been identified three Wnt genes, Wnts 10a,10b and 5a, whose expression is specifically upregulated in hair follicles at early morphogenetic stages. Of these, Wnt10b shows dramatic upregulation at the earliest stage of hair follicle development and is also specifically expressed in the postnatal hair follicles at the onset of a new cycle of hair growth. Furthermore, other reports showed that Wnt10b promoted the differentiation of primary skin epithelial cells toward hair shaft and inner root sheath of the hair follicle cells in vitro, elongation of the hair shaft in hair follicle cultures, and induction of hair follicle formation in the cultured embryonic skin tissue, whereas Wnt3a,5a, and 11 did not elicit such actions. Therefore,Wnt10b is strong candidate for the signals that cause the accumulation of nuclearβ-catenin in the epithelium and mesenchyme,and initiation of hair follicle development. Progress in this area of research may ultimately permit the development of novel therapies for the disorders of human hair follicle.In order to fully understand the exact function of Wnt10b on hair follicle development,we observed the morphogenesis and dynamic expression of Wnt10b/β3-catenin of the developing hair follicle in rat embryonic skin, in the meantime, a rat skin organ culture system was established that allowed synchronization of follicle formation and made the skin accessible to experimental manipulations. Then, an eukaryotic expression vectors containing 1170bp bp coding region of WntlOb gene was constructed and transfected into COS-7 cells to induce them to express Wnt10b protein. After that, to study the association between Wnt10b with the formation and distribution of placodes at the initiation of hair follicle development, we combined the skin organ culture system with both the recombinant Wnt10b protein and RNA interference, mimics transgenic gain of function and loss-of function approaches respectively.Chapter 1:Dynamic expression of Wnt10b/β-catenin during the morphogenesis of hair follicle in rat embryonic skinObjectiveTo investigate morphogenesis and dynamic expression of Wnt10b/β-catenin of the developing hair follicle in rat embryonic skin.MethodsThe dorsal skins of Sprague-Daivley(SD) rat aged embryo 13 day (E14) to postnatal 3 days (P3) were taken to paraffin embedding, section, HE staining, fluoroimmunoassay and immunohistochemical staining.ResultsIn the rat embryonic skin, the epithelium was composed of simple layer of epithelial cells at E13, then progressively differentiate and became multilayer; The hair follicle placode first appears at E15.5-E16.5,which morphogenesis almost completely finishs at postnatal 3 day; Wnt10b/β-catenin was uniformly expressed in the epithelium before the appearance of placodes and then eventually localized on the placodes when they occured. As morphogenesis continues, the expression of Wnt10b/β-catenin became restricted to follicular epithelial cells immediately overlying the dermal condensate and subsequently to matrix cells that contains precursors of the inner root sheath.ConclusionsThe first morphogenesis of hair follicle in SD rat dorsal skins probably begins at E15.5-E16.5 and finishs at P3; Wnt10b/β-catenin signal probably operates as a key epidermal message to regulate hair follicle morphogenesis.Chapter 2:Establishment of rat skin organ culture model to study the initiation of hair follicular morphogenesis and its correlative dynamic expression of Wnt10b/β-cateninObjectiveTo study the initiation of hair follicle morphogenesis, and observe the dynamic expression of Wnt10b/β-catenin in the developing hair follicle of skin organ culture model.MethodsThe dorsal skins of SD rat at embryos 14-17 (E14-E17) were cultured on a gelatin sponge-supported histoculture system at the air/liquid interface of DMEM with 10% fetal bovine serum(FBS) for 3-6 days, HE staining and fluoroimmunoassay were adopted. The dorsal skins removed from E15 were cultured in DMEM with FBS of different concentrations, the number of de novo hair follicle in the cultured embryonic skin was calculated and statistical analysis in the third day.ResultsThe model we established allowed skin tissues isolated from E14.5-E15.5 rat to develop in a manner that was histologically and temporarily similar to the process in vivo. However, the developing hair follicle ceased to continue when their morphogenetic process reached the forth stage, and the concentration of FBS did not show any significant effect on the development of hair follicle; Expression of Wnt10b/β-catenin was induced in culture, as was in vivo, but grew weak till it disappeared in culture for 6 days, which was accompanied by the development halt of the hair follicle.ConclusionsWe established a rat skin organ culture model in which the morphogenesis of hair follicle took place in a manner similar to in vivo, there is probably a close connection between Wnt10b/β-catenin and the early morphogenesis of hair follicle.Chapter 3:Preparation of rWntlOb protein and its promotion of morphogenesis of hair follicle in cultured rat embryonic skin Part 1:Construction of eukaryotic expression vectors of human WntlOb gene and its expression in COS-7 cellsObjectiveTo obtain eukaryotic expression vector containing the coding region of human Wnt10b, transfect the vector into COS-7 cells and detect whether it express WntlOb protein.Methods1170bp cDNA fragment was amplified from pUC19-Wnt10b by PCR and cloned into eukaryotic expression vector pEGFP-N1.The cloned insert was identified by double digestion of the recombinant plasmid with restriction enzymes EcoR I and Kpn I and direct sequencing. The recombinant plasmid was transfected into COS-7 cells with Lipofectamin M2000.The expression of WntlOb gene was detected by Western blotting and immunocytochemistry.ResultsEukaryotic expression vectors containing 1170bp bp coding region of WntlOb gene was constructed. COS-7 cells transfected with the recombinant plasmid expressed high level of Wnt10b protein in cytoplasm.Conclusion pEGFP-N1-WntlOb eukaryotic expression vector containing coding region of Wnt10b gene was constructed can provide a strong molecular tool for the studies of effect of Wnt10b gene.Part 2:Preliminary study on promotion of hair follicle development by Wnt10b protein in cultured rat embryonic skin and its underlying mechanismObjectiveTo further elucidate the promotion effect and possible mechanism of WntlOb on hair follicle development in vitro.MethodsThe dorsal skins of SD rat at embryos 14.5-15 were cultured in DMEM in the presence or absence of rWnt10b protein. WntlOb/β-catenin expression was analyzed by Western blot after two days, and the development of hair follicle was investigated in the third day.ResultsIn the presence of rWnt10b, The number of de novo hair follicle placodes in the cultured embryonic skin increased, which was accompanied by the upregulation of Wnt10b andβ-catenin proteins expression.ConclusionWnt10b protein is able to promote hair follicle development in the cultured embryonic skin, which may be achieved by the activation of canonical Wnt signalingChapter 4:Preliminary study on inhibition of the hair follicle development by siRNA targeting Wnt10b in the cultured rat embryonic skinObjectiveTo further investigate whether the suppression of WntlOb by RNA interference could prevent the formation of hair follicle in the cultured embryonic skin. MethodsWith the help of Guang Zhou Ribobio Co.,Ltd, we obtained three positive-sense and anti-sense strands of siRNA-WntlOb molecules that was synthesized by chemosynthesis method. The dorsal skins of SD rat at embryos 14.5-15 were cultured in DMEM in the presence of different small interfering RNA targeted to Wnt10b. Wnt10b/β-catenin expression was analyzed by real-time PCR everyday and by Western blot in the third day. The cultured embryonic skins were taken to paraffin embedding, section, HE staining in the third day, in which the number of de novo hair follicle was calculated and statistical analysis.Results Wnt10b gene in the cultured embryonic skin could be knockdown with the siRNA-based method;β-catenin mRNA was not greatly influenced by the downregulation of Wnt10b mRNA. In the presence of small interfering RNA targeted to WntlOb, the number of de novo hair follicle placode in cultured embryonic skin decreased,accompanied by the downregulation of Wnt10b andβ-catenin proteins expression in the third cultured day.ConclusionOur results suggest that in embryonic skin organ culture is a powerful method for analyzing the development of hair follicle. The downregulation of WntlOb mRNA and protein by siRNA reduces the number of de novo hair follicle placode. Wnt10b may control cytoplasmβ-catenin concentration at the protein level.
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