| The feathers of birds are derived from the feather follicle,which has multiple roles in insulation,homeostasis and aiding flight,and their development and cyclic cycle are the result of the interaction between epidermal and dermal cells,regulated by a complex combination of signals and factors.At present,most of the research on hair follicles is focused on mammals,but less on geese,which have a high down value.As an important economic trait and genetic improvement target in goose breeding,traditional breeding methods have been slow in progress.If the regulatory mechanism of the development of goose follicles can be explored and understood at the molecular and cellular levels,it will help to accelerate the progress of molecular breeding for goose down traits and bring new hope for goose breeding.The TGF-βand Wnt signalling pathways are known to be important regulatory pathways affecting the development of hair follicles,but their regulatory mechanisms in the development of feather follicles in poultry are not yet clear.Firstly,this study screened out the signaling pathways that may have important significance in the development of goose skin feather follicles through transcriptome sequencing and biogenesis analysis;subsequently,we focused on the expression patterns of TGF-β/Smad and Wnt/β-catenin signalling pathways during embryonic feather follicle development in Jilin white geese.Finally,the TGF-β pathway activator was used to investigate the effect of activating the TGF-β/Smad signalling pathway on skin fibroblast proliferation and apoptosis and hair follicle development,in order to speculate on its "role" in the development of avian feather follicles.The study is as follows.(1)Transcriptomic study of skin follicle tissue at key time points in embryonic follicle development in Jilin white geese.Jilin white geese were sampled at three feather hair follicle developmental stages: primary folliculogenesis(day 14/E14),secondary folliculogenesis(day18/E18)and primary follicle maturation(day 28/E28).Transcriptomic sequencing was performed by RNA-Seq technology on the dorsal skin follicle tissue of goose embryos at each stage.The results showed that 1280,5652 and 4733 differentially expressed genes were harvested in E18 vs E14,E28 vs E14 and E28 vs E18,respectively.KEGG analysis showed that they were highly enriched in the Wnt signaling pathway.GSEA enrichment analysis of sequencing data showed that TGF-β pathway was significantly enriched during feather follicle development.(2)Morphological observation of hair follicles in the dorsal skin of Jilin white geese and gene expression studies of TGF-β/Smad and Wnt/β-catenin signalling pathways.Samples were taken at four time points during embryonic development: early embryonic development(E14),middle embryonic development(E18),late embryonic development(E23)and late embryonic development(E28).The expression of TGF-β/Smad and Wnt/β-catenin signalling pathway genes was then quantified and localised with the aid of real-time fluorescence quantitative PCR,Western Blotting and immunohistochemistry.It was observed that feather buds appeared on the dorsal surface of the goslings at E14,transformed into classical hair follicle structures at E18 and fully matured by E28.In addition,collagen fibres appeared in the middle of embryonic development and were widely distributed throughout the dermis by the end of embryonic development;myofibres appeared in the late embryonic development and were mainly concentrated in the root sheaths inside and outside the feather follicles.Quantitative and localization analysis of genes related to the TGF-β/Smad and Wnt/β-catenin signalling pathways showed that the phosphorylation level of Smad3,a transcription factor downstream of the TGF-β/Smad signalling pathway,was significantly increased at E18 and E28(P<0.05),i.e.the activity of the TGF-β/Smad signalling pathway was significantly increased at mid-and end-stage.The expression of Wnt/β-catenin signaling pathway transcription factor LEF-1 was significantly increased at E28(P<0.05),indicating that the activity of Wnt/β-catenin signaling pathway was significantly increased at the end of embryonic development.Immunohistochemical staining revealed that the TGF-β/Smad and Wnt/β-catenin signalling pathways were prevalent in epidermal and dermal tissues in the pre-embryonic development,and gradually concentrated in the root sheath sites inside and outside the feather follicle as the embryo developed.(3)Investigation of the effect of TGF-β/Smad signaling pathway on the proliferation and apoptosis of goose embryo skin fibroblasts and feather follicle development.Goose embryo dermal fibroblasts were extracted and four groups of TGF-β/Smad signalling pathway activator TMAO were added at 0μM,low(3μM),medium(30μM)and high(300μM)concentrations to investigate the protein expression levels of genes related to the proliferation and apoptosis pathway by Western Blotting.The results showed that activation of the TGF-β signalling pathway inhibited the proliferation of skin fibroblasts and that the 3 μM activator addition group promoted their apoptosis.Using the embryo injection technique,50 mg/KG of TMAO was injected into the yolk sac of embryonic eggs at embryonic day 9(E9),and activation of the TGF-β signalling pathway significantly inhibited the growth of feather follicles and the elongation of the feather shaft when comparing the diameter of the dorsal feather follicles and the length of the feather shaft in goose embryos(P < 0.001).In conclusion,the expression of TGF-β/Smad and Wnt/β-catenin signalling pathways in the developing dorsal skin of the goose embryo is dynamic and regular,and they may involved in the regulation of skin cell proliferation and apoptosis and the development of feather follicle morphology and structure.The activation of the TGF-β/Smad signalling pathway may regulate the microenvironment of the feather hair follicle by mediating the proliferation and apoptosis of dermal fibroblasts,thereby affecting hair follicle development. |
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