The Mechanism Of Regulating Feather Follicle Development And Feather Growth By Wnt/β-catenin Signaling Pathway In Yellow-Feathered Chickens

The yellow-feathered chicken is an indigenous breed in China.The growth and development of feathers is an important commodity trait of the chickens.However,nearly15%of yellow-feathered chickens have different degrees of feather development problems in the large-scale poultry production.Such problem has seriously affected the appearance of the living bodies and the quality of the carcasses.However,the reason is still unclear.The objective of this study was to explore the growth and development of feathers in yellow-feathered chickens during the embryonic and post-hatch periods,and to explore the underlying relationship between the morphogenesis of feather follicles and Wnt/β-catenin signaling pathway.Based on the chick embryo model,we investigated the effect of Dickkopf-1（DKK1）,a specific inhibitor of Wnt/β-catenin signaling pathway,on the development of feather follicles and feather growth.We aimed to elucidate the mechanism of regulating the growth and development of feathers in yellow-feathered broilers.This study is mainly divided into three parts as following:Experiment 1 was conducted to investigate the growth and development of feathers in yellow-feathered broilers during the embryonic and post-hatch periods.During the embryonic period,the feather mass and length of 20 embryos were determined on embryonic（E）days 8,9,10,11,12,13,15,17,19,and the day of hatching（DOH）,respectively,to fit growth curves and describe the patterns of feather growth and development.After hatching,the feathers of 20 chickens on DOH,day（D）7,14,21,35,49,63,77,91,105,and 119,respectively,were collected to identify which growth model accurately described the feather growth pattern.Experiment 2 was conducted to investigate the morphogenesis of feather follicles in embryonic stage of yellow-feathered broilers,and to determinted the proteins expression of Wnt/β-catenin pathway.The back skin tissue of twelve embryos were collected on E7,8,9,10,11,12,13,15,17,19 and DOH,respectively.The morphogenesis of feather follicles were determined by H&E staining,and the density of feather follicles were measured.While the proteins expression of Wnt/β-catenin signaling pathway were detected by Western blotting and immunofluorescence.Experiment 3 was conducted to investigate the effect of in ovo injection of DKK1 on feather follicle development and feather growth of yellow-feathered broilers and its regulation mechanism.A total of 120 fertilized embryo eggs were randomly divided into four treatments.At E9,treatment 1 was not injected with any solution（control group,CON）,treatment 2 injected with 100μL PBS/egg（PBS group）,treatment 3 injected 100μL PBS/egg,containing 600 ng DKK1/egg（600 ng DKK1 group）,and treatment 4 injected 100μL PBS/egg,containing 1,200 ng DKK1/egg（1,200 ng DKK1 group）.After that,the eggs were transferred to incubator.The hatchability of fertilized eggs was recorded at DOH,feather samples were collected at DOH to determine feather mass.And the skin tissue samples were collected to observe the feather follicle development by H&E staining,and measured the density and diameter of feather follicles.Meanwhile,the proteins expression of Wnt/β-catenin signaling pathway were detected by Western blotting and immunofluorescence.The results showed that:Experiment 1,embryos began to grow feathers around at E10,and feathers grew rapidly during E13-E17.There was little change for the feather growth during E17-DOH.During embryonic period,the Gompertz function(Y=798.48e^{-203431.24exp（-0.87t）},R²=0.997,RSS=3355.511)provided the best fit of feather growth curve compared with Logistic and Bertalanffy models.After hatching,the mass and length of feather changed little from DOH to D14,grew rapidly from D21 to D91,and then grew slowly after D91.The three nonlinear functions can overall fit the feather growth curve well（R²>0.970）,and the Bertalanffy model(Y=116.88*(1-0.86e^-0.02t)³,R²=0.987,RSS=101.760)showed the highest degree of fit.Experiment 2,the skin appeared protrusion during E7-E9,feather buds began to form and gradually extension during E9-E11,epidermal cells rapidly aggregated and invagination formed feather follicle wall during E12-E13,and feather follicle morphology was basically completed at E15.Primary feather follicles appeared at E12,and gradually increased during E12-E15,then gradually decreased.The secondary feather follicles appeared at E15,and gradually increased during E15-DOH.The protein expressions ofβ-catenin significantly increased from E8 to E12（P<0.05）,then gradually decreased from E13 to DOH（P<0.05）.The expression patterns of the downstream target proteins of TCF4,Cyclin D1 and c-Myc were similar with theβ-catenin.The immunofluorescence result ofβ-catenin was consistent with the result of Western blotting assay.Experiment 3,compared with the CON and PBS group,injection of DKK1 into the yolk sac of yellow-feathered broilers had no significantly effect on hatching rate and embryo weight,while significantly reduced the relative mass of feathers in whole body or in the part of belly,neck,wings,head,and legs（P<0.05）.Compared with the CON and PBS group,the density and diameter of feather follicles were significantly decreased by the injection of DKK1（P<0.05）.In addition,the results showed that the DKK1 injection significantly down-regulated the expression ofβ-catenin and its downstream target proteins of TCF4,Cyclin D1and c-Myc（P<0.05）.The immunofluorescence result ofβ-catenin was consistent with the result of Western blotting assay.It can be concluded that:（1）The feathers showed slow-fast-slow growth of yellow-feathered broilers,and the Gompertz model exhibited the best performance to fit feather growth during the embryonic period,while Bertalanffy can overall fit the feather growth curve well during the growth period.（2）The feather follicles gradually formed during E7-E15,and the proteins expression of Wnt/β-catenin signaling pathway were significantly up-regulated during feather follicle morphogenesis.（3）In ovo injection of DKK1 significantly inhibited feather follicle development and feather growth by blocking Wnt/β-catenin signaling pathway in yellow-feathered broilers,indicated that the signaling pathway mediated the feather follicle development and feather growth of yellow-feathered broilers.