Study On The Mechanism Of BMP7 In Coat Color Formation

Coat color is an essential trait in vertebrate animals.Wool is an important raw material in the fiber industry,which has important economic value in some domestic animals.Presently,chemical dyes are mostly used to derive hair fibers of various colors,but many chemical dyes can cause allergies or even result in carcinogenesis.Thus,natural coat color is valuable to the fiber industry due to avoidance of potential harm to human beings caused by products generated from chemical dyeing.Therefore,many researchers have focused on developing natural animal coat colors.Some genes of BMPs can regulate the production or transfer of melanin and even the process of coat color formation.BMP6 is an important melanogenesis promoter in the BMP family.BMP7,a member of the same subfamily as BMP6,is one of the most biologically active and widely studied proteins in the BMP family.As an important member of the BMPs subfamily,BMP7 is upregulated in melanoma,and more importantly,it is expressed in mouse,rex,yak,goat,and sheep hair follicles,and it can regulate the function of cell,which related to pigmentation.Therefore,we hypothesized that BMP7 could regulate animal coat color production.We studied some genes expression of BMPs-SMADs signaling pathway in the skin of sheep with different coat colors.Then we applied research methods such as the construction of overexpression vectors,real-time quantitative PCR,western blot,and high-throughput sequencing to reveal the molecular mechanism of BMP7 in coat color formation.Results are as follows:1.We measured the melanin amount of back wool in white,brown and black Kazakh sheep.Results showed that the melanin amount showed a significant upward trend in white,brown and black wool.2.The expression and localization of BMP2,4,6,7 and its inhibitor Noggin were detected in the skin of sheep with different coat colors.qPCR and Western blot results showed that the expression levels of Noggin,BMP2,4,6,and 7 were increased in white,brown,and black sheep skin.IHC results showed that Noggin was expressed in the epidermis,root sheath,hair shaft,dermal papilla and hair matrix of sheep skin with different coat colors.BMP2 and BMP4 were expressed in epidermis and root sheath of sheep skin with different coat colors.BMP6 was expressed in the epidermis and root sheath of white Kazakh sheep.BMP6 was not only expressed in the epidermis and root sheath of brown and black sheep skin,but also expressed in the hair matrix.BMP7 was expressed in the epidermis,root sheath,and hair shaft of sheep skin with different coat colors.3.The expression and localization of BMPR1 B,BMPR2 and SMAD1/5 in white and black sheep skin.qPCR results showed that the expression levels of BMPR1 B,BMPR2,SMAD1 and SMAD5 mRNA were significantly higher in black sheep skin than those in white sheep.Western blot results showed that expression of BMPR1 B,BMPR2,and p-SMAD1/5 / SMAD1/5 in sheep skin were same as their mRNA expression.IHC results showed that BMPR1 B was expressed in epidermis,root sheath,dermal papilla and hair shaft of Kazakh sheep skin with different coat colors.BMPR2 and p SMAD1/5 were expressed in epidermis,root sheath,dermal papilla,hair matrix,and hair shaft of Kazakh sheep skin with different coat colors.4.We detected the effects of BMP7 in mouse melanocytes.The results showed that BMP7 promote the production of eumelanin amount,however,total melanin amount and phomelanin amount were decreased.Expression of melanin related gene(MITF,TYR,TRP1 and TRP2)and BMP7 receptor and its downstream genes(BMPR1A,ACVR1 A,SMAD1,and SMAD5)in the BMP7 group were higher than those in NC group.The growth of melanocytes was inhibited by BMP7.BMP7 could inhibite the cell cycle of melanocytes in the G2/M phase,and thereby inhibite the proliferation and migration of melanocytes.5.We detected the effect of BMP7 in mouse coat color formation.BMP7 was overexpressed and subcutaneously injected in mouse.The results showed that BMP7 could promote eumelanin amount formation and could promote the expression of MITF.6.Deep sequencing was performed in BMP7 transfection melanocytes to discover more mechanisms of pigmentation.The results showed that there were 46 genes which expressed differentially and related to coat color in NC group and BMP7 group.KOG classification analysis discovered that BMP7 regulated cell cycle control.qPCR analysis suggested that BMP7,Rnasek,Dnmt3 a,RSK2,SCAMP4,SLC38A1 and SLC39A9 mRNA expressed significantly difference between NC and BMP7 group,and these results were consistent with RNA sequencing results.7.We detected the expression and localization of RSK2 in sheep skin with different coat colors.qPCR and Western Blot showed that the expression of RSK2 mRNA and phosphorylated RSK2(pRSK2 / RSK2)in white sheep skin was significantly higher than that in black sheep skin.pRSK2 was located in the epidermis,root sheath,dermal papilla,and hair matrix of Kazakh sheep skin.In summary,BMP7 can regulate the growth of melanocytes and the production of melanin,and then affects the formation of coat color.BMP7 participates in multiple signal pathways in melanocytes,of which the BMP7-SMAD1/5 signaling pathway may regulate melanin production.RSK2 is also one of the potential regulated genes,which regulated by BMP7 in melanocytes.It provides strong evidence for exploring the mechanism of coat color formation.In addition,this study may provide novel insights for the treatment of pigmentation disorders and related diseases.