Screening And Identification Of Hair Follicle Initiation And Development Related Gene In The Fetal Period Of Inner Mongolian Cashmere Goat

Cashmere is an important economic product in Cashmere goat farming. The value of cashmere fleece is thought to be determined by the numbers and density of HFs. In recent years, molecular mechanisms that govern HF cyclic regeneration have been revealed in Cashmere goat breeds, but molecular mechanisms that govern HF initiation, growth and development process in the fetal period is relatively scarce. At present, life science has entered into the Post-Genome Era, the transcriptome and proteomics are important contents of functional genomics research, and also provide useful ways for further study on functional mechanism of hair follicle growth and development. In our study, skin samples at 10 different fetal periods were collected from the lateral body of each Cashmere goat. The RNA-seq and ITRAQ at three specific developmental stages:45-,55- and 65-day-old fetuses (E45, E55 and E65) were carried out in the present study according to the preliminary results, the results of sequencing were verified by qPCR and Western blot (WB) respectively. In order to enhance our understanding of the molecular regulation mechanism and provides research foundation for cashmere HF initiation and development, we carried out omics conjoint analysis. The main results were as follows:1. Through the transcriptome sequencing, each sample obtained average 11 G data, a total of 4581 differentially expressed genes (DEGs) were identified. Among which,1590,2442, and 3844 DEGs were identified in E55/E45, E65/E55, and E65/E45, respectively (.P<0.05). Of the 10 genes we randomly selected to evaluate for the validation rate,8 genes expression were consistent with RNA-seq.2.4581 genes showed differential expression patterns and were divided into 10 categories by k-means clustering, each category genes may play the similar roles in the process of hair follicle growth and development. The results of hierarchical clustering analysis of gene expression suggest that E55 and E45 have similar expression patterns. According to the results of GO enrichment analysis, DEGs were identified in the cellular component, molecular function and biological process with significant enrichment (P<0.05). KEGG enrichment analysis was further performed. Some DEGs were divided into in WNT, TGFβ and ECMR interaction pathways and the DEGs exist in these pathways may be acted as the key candidate genes in the process of hair follicle initiation and development. Through high-throughput sequencing, types of alternative splicing including A5SS, A3SS, ES and IR of the nine samples were analyzed and the results suggest IR is the most common type.3. The expression of SFRP4, WNT3, WNT10al and APC2 in different periods of embryonic skin was detected by real-time quantitative PCR. The results suggest that expression of these genes in E45, E55 and E65 were in line with RNA-seq. The results not only further verified the accuracy of RNA-seq, but also preliminary explored the functional mechanism of Wnt pathway related gene in the fetal period of Cashmere goat.4. ITRAQ analysis generated 1,172 proteins identified in nine skin samples from cashmere goats of E45, E55 and E65. The numbers of the differentially expressed proteins in E55 vs. E45, E65 vs. E55, and E65 vs. E45 were 143,176 and 201, respectively (P<0.05). The expression of Decorin in E45, E55 and E65 by using western blotting is consistent with the result of ITRAQ.5. Distribution of the proteins in different GO categories analysis found differentially expressed proteins may have a greater influence on cell cycle and signal transduction. Among subcategories during HF morphogenesis, cell and cell part were dominant in the main category of cellular component. Cellular process and binding were dominant in the main categories of biological process and molecular function. KEGG pathway analysis categorizes proteins functions by biochemical pathways. Some differentially expressed proteins were assigned to the ECMR interaction, Focal adhesion, PI3K-Akt, MAPK, TGFβ, VEGF, p53 and WNT signaling pathways.188 differentially expressed proteins (P<0.05) in three different hair follicle developmental stages were classified by Cluser 3.0, a heat map of 159 proteins illustrates three different profiles, among these proteins, sixty-three proteins were constantly increased, forty-five proteins were constantly decreased and fifty-one proteins showed a decreasing trend before they rise in the three developmental stages6. Omics conjoint analysis found that 238 genes were common up-regulated and 920 genes were common down-regulated between E55vs E45 and E65vs E45. E55vs E65 has 585 up-regulated genes and 1348 down-regulated genes compared with E45 vs E55. E55vs E65 has 69 up-regulated proteins and 96 down-regulated proteins compared with E45 vs E55. Among them, RNA-seq data of some genes such as DCN, KRT7 OXCT1, MYH10 and ANPEP is consistent with ITRAQ data in three fetal periods.7. mRNA and protein expression profiles of Decorin (DCN) in 10 different periods of embryonic skin was detected. The results suggeted that the mRNA and protein at E45, E55 and E65 were constantly increased, the expression in 75-,95-and 105-day-old embryos are higher than other fetal period, the expression in 115-,125-and 135-day-old embryos appeared a downward trend.8. In order to study the DCN mechanism of action in the cell level, we construct a overexpression vector pEGFP-CI/DCN.