T?4 Function During Cashmere Goat Hair Follicle Growth And Development

Cashmere goat is a precious livestock genetic resource in China,among which the Albas cashmere goat has become a world-class breed through long-term natural selection and artificial breeding.Its cashmere has been honoured as ‘soft gold' or ‘fibre gems' internationally.However,historically,due to the lack of scientific germplasm conservation awareness,the blind hybridization of local varieties with foreign varieties has led to degradation of the population resources.To further protect and develop the excellent germplasm resources,modern molecular breeding technology is urgently needed to breed high-quality and high-yield cashmere goats and to conserve and innovatively use the cashmere goat's germplasm.The growth and development of hair follicles(HF)have important effects on the yield and quality of cashmere.In this study,we have explored the function of T?4 during cashmere goat HF growth and development from three levels.1.Screening the target gene and studying its function in vitroTo identify key genes that affect HF growth and development in cashmere goats,we analysed the RNA sequence raw datasets of cashmere goat skin tissue and dermal papilla cells of secondary hair follicle(SHF-DPC)samples from HF anagen to telogen and identified a set of 80 differentially expressed genes(DEG)as a preliminary range of screening.Five candidate genes were identified by gene ontology(GO)analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis,and construction of protein-protein interaction network(PPI).Based on gene set enrichment analysis(GSEA)for the candidate genes,we ultimately chose to focus on thymosin ?4(T?4)after experimental verification.T?4,first isolated from bovine thymus,is a highly conserved G-actin-sequestering protein composed of 43 amino acid residues,with a molecular weight of 4.9 kDa.The molecule is involved in various biological processes,including promoting cell migration,angiogenesis,and wound healing,and plays a key role in mouse HF growth and development.However,the role of T?4 in cashmere goat HF growth and development remains unclear.To further analyse the effect of T?4 on cashmere goat HF growth and development in vitro,T?4 overexpression(T?4-OE)and RNA interference(RNAi)vectors were successfully constructed.SHF-DPC were cultured in vitro and identified.Then,the T?4-OE vector was transfected into SHF-DPC,and the cell proliferation was detected using EdU.The cell proliferation ability of the T?4-OE group was enhanced compared with the control(Ctr)group.When T?4(T?4-KD)was knocked down in SHF-DPC,the cell proliferation ability of the T?4-KD group was weakened compared with the Ctr group.Moreover,we restored the cell proliferation ability of the T?4-KD group by overexpressing T?4 in the cells.Therefore,it was confirmed that T?4 can promote the proliferation ability of SHF-DPC in cashmere goats.2.Generating T?4-gene-modified cashmere goats and studying the effect of T?4 on the cashmere trait in vivo in the parental generationTo generate HF-specific expression in T?4-modified cashmere goats,we first established the nuclear donor cell lines of T?4 overexpression driven by the HF-specific promoter KAP6.1.We then injected it into the oocytes using somatic cell nuclear transfer technology(SCNT).We obtained 1155 reconstructed embryos and transferred them to 241 recipient goats.Fifteen T?4-gene-modified cashmere goats with good growth status were obtained.Continuous monitoring indicated no significant difference between T?4-gene-modified cashmere goats and wild type(WT)in terms of physical index.In terms of cashmere index,the cashmere yield of T?4-gene-modified cashmere goats was significantly increased,up to 380 g,but there was no significant difference in cashmere thickness and wool length compared with WT.To further investigate the correlation between the T?4 gene and cashmere goat HF growth and development,T?4-gene-modified cashmere goats with significantly improved cashmere yield(T?4+ group)and with no significant increase in cashmere yield(T?4-group)were compared with the WT group.Southern blot showed that the T?4+ group and T?4-group genomes had more T?4 gene copy numbers than the WT group.qPCR and western blot showed that T?4 expression in the T?4+ group was higher than that in the WT group,but there was no significant difference between the T?4-and WT groups.This suggests that T?4 in the T?4-group was inhibited,resulting in gene silencing.Haematoxylin-eosin(H&E)staining of paraffin sections of skin tissue showed that the ratio of SHF to PHF in the T?4+ group was significantly higher than that in the WT group.The ratio in the T?4-group was not significantly different when compared with the WT group.Therefore,T?4-gene-modified cashmere goats can increase cashmere yield by increasing the number of SHF.To further investigate the effect of the internal mechanism of T?4 on HF growth and development and the cashmere trait,717 DEG and 177 proteins interacting with T?4 were identified through integrated transcriptomics and proteomics analysis.Based on the verification of EdU cell proliferation,keratin 4(KRT4)could interact with T?4 to affect HF growth and development and improve the cashmere trait.Finally,the potential downstream signalling pathway of the T?4-KRT4 axis was analysed.Compared with the WT group,ERK and phosphorylated ERK levels in the T?4+ group were significantly increased,while ERK and phosphorylated ERK levels in the T?4-group were not significantly different from those of the WT group.Thus,T?4 may interact with KRT4 to affect cashmere goat HF growth and development through the ERK signalling pathway,which lays a foundation for in-depth study of the molecular mechanism.3.Generating T?4-gene-modified offspring and studying the genetic stability of the cashmere trait in vivo of the first filial generationTo breed offspring with the excellent cashmere trait of T?4-gene-modified cashmere goats,23 T?4-gene-modified cashmere goat offspring were successfully established in two years by SCNT and natural mating(NM).Continuous monitoring showed that,in terms of physical index,the SCNT and NM groups were normal,with no significant difference from the WT group.In terms of cashmere index,compared with WT,the amount of cashmere produced by SCNT was increased,up to 320 g.There was no significant difference in cashmere thickness and fibre length between the NM and SCNT groups compared to WT.Overall,the genetic stability of the SCNT group was higher than that of the NM group.To further analyse the relationship between the genetic stability of the cashmere trait and the T?4 gene,the T?4 gene in the SCNT and NM groups was detected at different levels.Absolute quantitative PCR showed that both the SCNT and NM groups had more T?4 gene copies than the WT group.However,immunohistochemistry(IHC)showed that T?4 expression in the SCNT group was higher than in the WT group,and T?4 expression in the NM group was not significantly different from that in the WT group.The results of qPCR and western blot were mutually verified with IHC.The above results suggest that epigenetic modification of the T?4 gene was the main reason for the difference in genetic stability between generations with the SCNT and NM groups.To further study the effect of epigenetic modification on the genetic stability between generations,we performed integrated genome-wide methylation and transcriptome analysis;58 DEG,336 differentially methylated regions(DMR),and two genes driven by methylation between generations were identified.The enrichment of GO function in DMR and DEG indicated that the two results were highly consistent,suggesting that DMR and DEG affected the genetic stability of the intergenerational cashmere trait through the same method.The KEGG pathway enrichment analysis between DMR and DEG showed substantial differences,suggesting that DMR and DEG could affect intergenerational genetic stability through different signal pathways.Taken together,T?4 plays an important role during cashmere goat HF growth and development,and it has important theoretical and practical significance for improving the cashmere trait.