The Role And Mechanism Of BMP And FGF Signaling In The Development Of Hair Follicle In Mice

Mutations in numerous members of BMP signaling and FGF signaling pathways lead to several severe human syndromes.For example,dysfunction of fibroblast growth factor 8(FGF8)leads to idiopathic hypogonadotropic,holoprosencephaly,cleft lip and palate,etc.Patients with diopathic hypogonadotropic are accompanied by absence of axillary hair and pubic hair.BMP signaling and FGF signaling play crucial roles in the development of hair follicle.It has been reported that the expression of BMP2 is found to be significantly decreased in patients with alopecia areata,which may be one of the reasons for the onset of alopecia areata.In our study,we focused on the roles of Bmp2,Bmp4,Fgf8 and Fgf18 in the development of mouse hair follicles(HFs).It is well-known that,Bmp2 and Bmp4 are very important ligands in the BMP pathway,which play an irreplaceable role in the organogenesis,development and differentiation.The expression of Bmp2 and Bmp4 were detected in the initial morphogenesis of HFs.Interestingly,their expression patterns changed continuously with the growth cycle of HFs.In this paper,we generated mice carrying Bmp2 specific deletion in epithelial cells(epidermis)using floxed Bmp2 and K14-Cre alleles to study the role of BMP2 in the development of HFs.K14-Cre;Bmp2^f/f mice can survive to adulthood without obvious phenotypes.However,its hair began to fall off periodically from the 19th day after birth.To further determine whether Bmp2 is involved in regulating the development of early hair follicles,we collected embryos during the critical period of hair follicle development and performed histological examination.HE staining showed that K14-Cre;Bmp2^f/f mice did not exhibit obvious abnormalities during the early morphogenesis of HFs.The results of immunohistochemistry and in situ hybridization also showed that the expression of marker genes in early hair follicle development including Lef1,Shh,Bmp4,and Sox2remained unaltered in the HF of K14-Cre;Bmp2^f/fmice at E14.5 and E17.5,as compared to controls.The Hematoxylin/eosin staining results further suggesting that delation of Bmp2 shortened catagen and telogen phase in mutant mice.Scanning electron microscopy and semi-thin sectioning showed that delation of Bmp2 resulted in malformation of cuticle of the hair shaft.The results of transmission electron microscopy further confirmed that the high-density electron layer of hair shaft in K14-Cre;Bmp2^f/fmice was disrupted.The results of immunohistochemistry and immunofluorescence revealed a significant decrease in the expression of K82 and AE13.The above results indicated that BMP2 signaling in epidermal cells is an essential growth factor for regulating postnatal hair follicle development in mice,and conditional knockdown of Bmp2 in epidermal cells leads to disruption of the hair follicle development cycle and abnormal cuticle of the hair shaft,ultimately leading to cyclic alopecia in K14-Cre;Bmp2^f/fmice after birth.Next,we crossed Bmp4^f/fmice and K14-Cre mice to generate K14-Cre;Bmp4^f/fmice carrying Bmp4 specific deletion in epidermal cells.Unexpectedly,the growth of hair appeared identical between WT and K14-Cre;Bmp4^f/fmice.To investigated whether Bmp4 play a role in hair follicles development,we performed histological analysis of K14-Cre;Bmp4^f/fmice and their WT littermates.A series of experiments were performed to compare the morphological difference and expression of key genes in the development of hair follicle in wild-type mice and K14-Cre;Bmp4^f/fmice,including Lef1,Shh,Sox2,AE13,AE15,Gata3.These results suggested that although weak BMP4 signal was detected in hair follicle epidermal cells at the beginning of hair follicle development,BMP4 signal in epidermal cells have little role in regulating the development of hair follicles,or its function could be compensated by other ligands from BMP family.Therefore,the development of hair follicles was normal in K14-Cre;Bmp4^f/f mice.To futher confirm whether Bmp4 has no effect on hair follicle development.We crossed K14-Cre mice,Bmp2^f/f mice and Bmp4^f/f mice to generate K14-Cre;Bmp2^f/f;Bmp4^f/f mice.The K14-Cre;Bmp2^f/f;Bmp4^f/f carrying Bmp2 and Bmp4 specific deletion in epithelial-derived epidermal cells.We found that K14-Cre;Bmp2^f/f;Bmp4^f/f mice began to lose their hair at P13.K14-Cre;Bmp2^f/f;Bmp4^f/f mice could continue to maintain a hairless state was similar to nude mice until a new wave of hair emerged,but the newly erupted hair was very sparse.The results of immunofluorescence confirmed that Bmp2and Bmp4 regulate the development of hair follicles and differentiation of the inner root sheath cuticle jointly.Given the critical role of Fgf8 in embryonic development,we generated a mouse model that overexpresses Fgf8 specifically in the epidermis.Interestingly,these mutant mice exhibited stunted,smaller bodies and severe hypotrichosis.Histological analysis showed that the hair follicles in the mutants were arrested at stage 2 of hair development.The density of hair follicles in the mutant mice was also lower compared to that in the control mice.The experimental results of in situ hybridization showed that the continuous activation of Fgf8 increased the expression of Bmp4,thereby reducing the density of hair follicles.The results of immunohistochemistry showed that overactivation of Fgf8reduces the expression of Shh and Gli1,while decreasing Shh expression inhibits the downward growth of hair follicles.Although the expression level of Lef1 in mutant mice is not significantly different from that in wild-type mice,there are obvious abnormalities in its expression position in mutant mice.Compared to wild type mice,the expression of Sox2 in the hair follicles of mutant mice was similar to WT mice.The results of immunofluorescence confirmed that excessive activation of Fgf8 would increase PDGF signal.Overexpression of Fgf8 inhibited the proliferation of epidermal cells and simultaneously promoted apoptosis,leading to the arrest of hair follicle development.To summarize,our study demonstrates that disordered FGF8 signaling plays an important role in the regulation of hair follicle development.It has been reported that the delation of Fgf18 shorten the telegen phase of hair follicles.Given the critical role of Fgf18 in hair follicle development,we generated a mouse model that overexpresses Fgf18 specifically in the epidermis to further study the role of Fgf18 in hair follicle development.Interestingly,these mutant mice showed abnormal hair follicle development.The histological analysis showed that the development of hair follicles in mutant mice was delayed and the growth rate of hair follicles was decreased from the neck to the tail.The results of alkaline phosphatase staining showed that the density of hair follicles in mutant mice was significantly reduced compared with wild type mice.Immunohistochemical results showed that the expression of Lef1 and Sox2 was not significantly different between mutant and wild-type mouse hair follicles at E14.5 and E17.5.The expression of Shh in the hair follicles of mutant mice was significantly reduced.The results of immunofluorescence showed that the expression of Pdgfr?in the hair follicles of mutant mice and wild-type mice were similar.Overexpression of Fgf18 promotes epidermal cell apoptosis.To summarize,our study demonstrates that FGF18 signaling plays an important role in the regulation of hair follicle development.In summary,this paper focuses on the role of Bmp2,Bmp4,Fgf8 and Fgf18 in the development of hair follicles.Our study illustrates cyclic alopecia of mice caused by Bmp2 mutations,highlights the crucial role of Bmp2 in hair follicle development,and emphasized that Bmp2 and Bmp4 play a crucial role in regulating the development of inner root sheath cuticle.Both Fgf8 and Fgf18 overactivation can trigger abnormal hair follicle development by reduction of the number of hair follicles and delaying hair follicle development.During the development of hair follicles,FGF and BMP form a complex signal network regulates hair follicle development.