Morphological analysis of goat skin cycle changes
We observed the skin tissue of cashmere goats by tissue sectioning technology. As the results showed (Fig. 1), The number of secondary hair follicles in cashmere goats decreased gradually from December to March (Fig. 1 L, A, B and C). The lowest value was reached in March (Fig. 1 C), and the statistical values of each trait were also lower. The division and the extension to the dermis of hair follicles began in April (Fig. 1 D), The statistics of secondary hair follicles also began to increase at the same time. It is observed that there is a velvet appearance on July (Fig. 1 G). Most villi grow out of body surface from August to September (Fig. 1 H, I). At the same time, the statistical value of secondary follicles reached the highest level, This period is considered to be the peak period of villous growth. In October, hair follicle bulb cells began to enlarge, gradually aged and died, and the dermal papillae began to atrophy, The statistics of secondary hair follicles gradually decreased (Fig. 1 J). In December, the hair follicle roots rose to the sebaceous glands, and the secondary follicle statistics reached the lowest level (Fig. 1 L), This state has been maintained until February of next year. So we made the initial inference that The cycle of secondary hair follicles in cashmere goats can be divided into growth period from March to September, resting period from September to December and regression period from December to March. Generally speaking, we can find that cashmere hair cycle can be divided into three periods by observing the skin tissue morphology, but the key points of each time can not be determined, which needs further study.
Gene expression difference analysis
We first screened and analysed the data quality (Table 1) and data length distribution (Table 2).Then we compared the skin transcriptome data of cashmere goats in 12 months with each two seats (Fig. 2). It was found that the number of differentially expressed genes was the largest between February and March. The total number of differential genes was 1059, of which 219 were up-regulated and 840 were down regulated. In March and April, the number of differentially expressed genes was 731, of which 550 were up-regulated and 181 were down regulated. In June and July, there were 418 differentially expressed genes, of which 388 were up-regulated and 30 were down regulated. Those results showed that the expression of genes was initially up-regulated or down-regulated during the initiation of secondary hair follicle growth. Along with the advance of hair follicle initiation, the number of down-regulated genes began to decrease, and the number of up-regulated genes continued to increase. After the completion of the initiation process, the gene changes tended to be stable. The results further showed that the hair follicle development was initiated by the combination of up-regulation and down-regulation of genes in the early stage of initiation, and the gene expression returned to normal level after initiation. Comparing the data between June and July, we found that there was another significant change in gene expression during villus outgrowth. We believe that this change promotes villi to grow out of the body surface, but whether there are other roles remains to be further studied. From August to February of the next year, the secondary hair follicles genes expression changed significantly from quiescence to degeneration. This results further showed that the initiation of secondary hair follicles in cashmere goats began in March. Finally, it is worth mentioning that the number of differentially expressed genes increases first and then decreases from February to March and then to April, thus proving again that the secondary follicle cycle starts in March.
Classification of gene function annotation
According to the GO classification statistics, the skin expression genes can be divided into three main categories: biological functions, cell components and molecular functions. In this study, 51078 transcripts were noted with GO annotation. Among them, in biological function, the most annotated transcript is cellular process. In cellular component, most of the transcripts have been transcribed to cell and cell part. In molecular function, most of the transcripts have been transcribed to binding. It is speculated that during the hair follicle cycle, the changes of gene expression lead to the changes of the number and state of cells in the hair follicle structure, which further leads to the occurrence or shedding of villi.
Group clustering analysis of natural periodic samples
In order to further explore the rule of gene expression, We calculated the correlation coefficient and cluster analysis of all gene expression levels in the 12 month natural cycle (Fig. 4). The results show that clustering information can be divided into three categories. The sample LZH3 was isolated because of the great changes in the gene expression of the follicle promoter. The sample LZH2-LZH7 ware considered to be the initiation process of hair follicle growth. Sample LZH8-LZH12 ware clustered together because it is thought to be the process of secondary hair follicles from vigorous growth to recession. Gene expression remained relatively unchanged from December to March, so the clustered sites were at the end of degeneration and before growth, They were considered to be the resting period of hair follicle development. Combined with previous studies in this article, we found that there ware several critical periods in the division of secondary hair follicle cycle. March is considered the key point for hair follicle cycle to start, September is the key period for the vigorous period of the hair follicle cycle and the beginning of the recession, December is the key point for the end of the hair follicle recession and the beginning of the rest period, These three critical periods determine the key signals in hair follicle and villi growth.
Extraction and analysis of target gene expression information
In order to explore the expression patterns of genes that play a key role in the cycle, we extract the expression information of all target genes for 1-12 months, and then cluster the expression patterns by analysis and exclusion. Gene expression patterns of several pathways related to villus cycle were obtained (Fig. 5A). The results showed that the expression pattern of genes related to villus cycle was consistent with our analysis of differential gene expression. The results further supported that villus of villus cycle began to enter the start stage in March, entered the regression stage in September and entered the end stage in December. However, the development process of villus cycle can not be visualized through the skin appearance results, because the development of cycle precedes the growth of villus, and there is a causal relationship between them. The period of villus growth is visually observed by tissue sections, and there is a direct relationship between villus growth and the expression of keratin. Therefore, in order to further verify the villus growth cycle, we also clustered the expression patterns of keratin and keratin-related genes (Fig. 5B). The results showed that the expression of keratin was consistent with the results of tissue sections, which further supported that the villus cycle first started (degenerated or rested), and then cascaded leading to changes in the expression of keratin gene, thus promoting the occurrence of villus (growth or degeneration). In order to further study the relationship between keratin gene and cycle, we take the gene with the highest expression as a case for further study.
QPCR analysis of key genes
Among keratin and keratin associated proteins, keratin associated protein 3-1 ranked first in cluster 11 and the expression level was 80824 at the growth stage and 23856 at rest stage. The expression of keratin associated protein 3-1 in cashmere for 12 months ware confirmed by quantitative PCR (Fig. 6 A). The results showed that KAP 3-1 was expressed in the skin at different stages of a year, but the expression of KAP 3-1 was significantly different (P<0.05) and fluctuated periodically in 12 months. The expression level of three months in August, September and October was significantly higher than that in other months (P < 0.05). Combined with previous studies, the expression quantity was verified by dividing different periods (Fig. 6 B). It was found that the expression of KAP3-1 gene in growth phase was significantly higher than that in rest or regression phase. Subsequently, To verify the stability of gene expression. We examined the expression of two other genes KAP 8-1 and KAP 24-1 in cashmere goat skin at several stages by fluorescence quantitative analysis (Fig. 7). The relative expression of KRTAP 8-1(Fig. 7A) and KRTAP 24-1(Fig. 7B) genes in Inner Mongolia cashmere goats' skin showed periodic variation, which was consistent with the hair follicle development cycle. It is indicated that KRTAP 8-1 and KRTAP 24-1 genes played a positive role in controlling cashmere wool growth and closely related to the regulation of villi growth and cycle transformation.