Fetal samples from the body side skin of cashmere goat embryos were collected at the Aerbasi White Cashmere Goat Breeding Farm (Inner Mongolia, China) at 10 time points of fetal age (45, 55, 65, 75, 85, 95, 105, 115, 125, and 135 days). Samples were collected from 3 fetuses for each of these time points. For the collection of adulthood samples, three adult sheep were selected from the Inner Mongolia Cashmere Goat Breeding Farm, and skin samples were collected from the lateral body. Samples were collected every month on the same day, and samples were collected continuously for 12 months; Each time approximately 1 cm² of the skin collecting. All above-mentioned samples were cleaned with diethylpyrocarbonate water, numbered, quickly transferred into liquid nitrogen for freezing, and stored in a refrigerator at −80°C for future use. In this experiment, the breeding environment was in compliance with the standards relevant to an ordinary animal laboratory facility in China National Standard “Laboratory animal environment and facilities” (GB14925-2010). The feeding of and the experimental operations on animals were in accordance with the animal welfare requirements. The total RNA and proteins were extracted from each skin sample for later use.

Early in the developmental process of the formation of the structure of cashmere goat hair follicle, we conducted a morphogenesis study and found that on gestational day 45 fetal epidermis was the only layer of flat cells that did not form primary follicles [11]; Inner Mongolia Cashmere Goats develop the complete structure of their epidermis from gestational day 45 to gestational day 55, the primary follicles have not yet begun to develop during this period. Keratinocytes were aligned neatly on epidermal base (Figure 1A). During the development of the fetus from day 55 to day 65, the hair buds increase in length significantly, penetrating into the cells of the dermis. The secondary follicle primordium can be observed from the fetal side of the body (Figure 1B); During the development of the fetus from day 65 to day 75, the secondary follicle primordium can be observed from the fetal side of the body, secondary follicles begin to occur (Figure 1C). Most secondary follicles develop their complete structure by the 125th day of fetal growth, and hair matrix cells surround the dermal papillae. Both dermal papillae and primary hair follicles have an oval shape which facilitates the penetration of villi through the body surface (Figure 1D). On day 135, a part of the hair follicles are mature, and the villi have pierced through the body surface [10] (Figure 1E). At birth, all primary follicles are mature, whereas only a small number of secondary follicles are mature at that stage. Within 18 months after birth, increasingly more secondary follicles mature, and at the age of six months, hair fibers have emerged from all secondary follicles, meaning that only the secondary follicles finish their development six months after birth. The growth pattern of cashmere wool showed a strong seasonal variation of adult. In a cycle, hair follicle activity includes anagen, catagen, and telogen [12] (Figure 2).

Comparison was conducted between the gestational samples collected on days 55 and day 45, day 65 and day 45, and day 55 and day 65. We established the presence of up to 2,847 differentially expressed genes (p≤0.05). The overlap analysis of these differences in the gene expression of the three parts (45 vs 55, 45 vs 65, 55 vs 65) resulted in the generation of a total of 204 expressed genes (Figure 3). The results of the cluster analysis performed in nine of the samples show that most of the samples cluster together according to the development period of hair follicles with only a few exceptions (Figure 4).

Basing on the evidence of some recent reports of studies on hair follicle development at home and abroad, we hypothesized that these three genes of the Fox gene family (FoxI3, FoxN1, and FoxE1) are probably involved in the growth and development of the follicle and the formation of the hair. Of them, FoxI3 and FoxN1 were included in the 204 differentially expressed genes that were common in all three periods. Therefore, FoxI3, FoxN1, and FoxE1 were selected as candidate genes for conducting research on their expression level in skin follicles.

Relative expression level of FoxN1, FoxE1, and FoxI3 genes in cashmere goat fetal skin hair follicles: The genes FoxN1, FoxE1, and FoxI3 were expressed in the skin tissue of cashmere goat fetus in all 10 experimental time points (Figure 5). The expression level of gene FoxN1 did not change significantly (p>0.05) from day 45 to day 95, but there was a significant increase in its expression level from the beginning of day 95 (p<0.05). The peak expression level occurred on day 135, whereas the expression level on day 115 was slightly lower, but both of them were significantly higher than the expression levels of the other fetal periods (p<0.05). The level of expression of FoxE1 gene showed an overall upward trend, although there was no significant change in the expression level from day 45 to day 85 (p>0.05). The expression level on day 95 was relatively higher than those at other time points of fetal age, but it reached its peak value on day 135, which was significantly higher than those of the other fetal development time points (p<0.05). The trend for FoxI3 gene expression in fetal skin tissue of cashmere goat in the 10 time points, with a peak on day 75, was opposite to the trend observed for FoxN1 and FoxE1. The gene expression level on day 85 was lower than that on day 75 but was still significantly higher than that of days 45, 55, and 65 (p<0.05). The expression level of gene FoxI3 in the hair follicles of the skin on day 65 was significantly higher than that on day 45 (p<0.05).

Venn diagram analysis was conducted on the continuously up-regulated genes in the fetal skin samples of cashmere goats on days 45, 55, and 65 (Figure 3). The comparative gene annotations on day 55 vs day 45, day 65 vs day 45, and day 65 vs day 55, revealed the presence of a total of 204 genes. This paper focuses on FoxN1 and FoxI3 was contained in these 204 genes. The cluster analysis of the differentially expressed genes in the different periods (Figure 4) showed that biological repeatability is the best in 65 days. On 45 and 55 days clusters of two samples were formed, which means the gathered samples were reliable. As can be seen from Figure 2, some genes showed a trend of up-regulation at these three time points, and FoxN1 and FoxI3 were two of them. In an earlier study FoxN1 was mainly expressed in the follicular epithelial cells and the skin hair follicle [8]. Other studies showed that patients suffering FoxE1 mutations had defective hair follicles, which suggests that FoxE1 may be involved in hair follicle morphogenesis. Therefore, the candidate genes FoxN1, FoxI3, and FoxE1 in the hair follicles of cashmere goats were selected for the experiments in our research.

According to previous research reports that Inner Mongolia cashmere goats develop the complete structure of their epidermis from gestational day 45 to gestational day 55. Nevertheless, the primary follicles have not yet begun to develop during this period. At the age of 55 days to 65 days, the formation of primary follicle. The formation of secondary follicles begins from gestational day 65 to day 75. Then, the primary follicles on the body side grow fast from gestational day 95 to gestational day 115. In addition, the secondary follicles on the body side also grow fast from gestational day 105 to gestational day 125. During the process of changes of follicle cycling, the upper section of hair follicles, i.e., the sebaceous glands and the part above the bulge remain stable, and apoptosis and regeneration do not occur. The lower section of the follicles, namely the hair follicles and the hair bulb part that is below the bulge, experiences morphological changes, such as growth, anaplasia, and diapause. Nonetheless, the changing patterns of the primary and secondary follicles in cashmere goat are quite different and dependent on the seasonal changes. No considerable changes are observed in the traits of the primary follicles as most of them remain active, and only a part enter the catagen phase. However, the secondary follicles show strong regularity that is based on the seasonal light cycle. From April to November, the secondary follicles gradually enter a period of growth and then of catagen (from December to January), before entering the telogen phase (from February to March). The stem cells are activated during the growth period, and the cells near the dermal papilla begin proliferating, forming new hair bulbs. After the formation of hair balls, the inner root sheath and the hair shaft begin to differentiate. It is in this period that the hair follicles begin to grow, and hair balls proliferate rapidly, and the duration of the growth period determines the length of the hair shaft. However, the inner root sheath then degenerates at the funnel part. Importantly, extensive apoptosis occurs in the catagen phase, and the differentiation is stopped. During this period, the hair bulbs become wider and larger, the hair roots go up, the claviform hair is formed, and the dermal papillae get in contact with the epithelia. In the telogen phase, the clavellate hair is anchored, apoptosis is terminated, the dermal papillae are connected with the bulge stem cell niches, and the hair follicles become dormant and enter the telogen stage. In this period, there is no significant proliferation, apoptosis, and differentiation, and the stem cells are located near the dermal papillae [13,14]. These processes are regulated by a series of signaling molecules, including bone morphogenetic protein (BMP) family, homeobox genes, fibroblast growth factor-5 (FGF5), that are needed to elicit the starting signal, the signal to maintain the growth of hair follicles, and the one initiating inhibition of the hair follicle growth. According to the evidence presented in reports published recently, the major upstream regulatory signaling pathways of FoxN1 are the Wnt pathway and the BMP pathway. It is speculated that the multifunctional developmental regulator BMP-4 may be used as a signaling molecule to induce the expression of FoxN1. Earlier studies in mice revealed that both the first layer of skin basal cells and some of the epidermal basal cells express FoxN1, whereas the starting signal for initiation of the development of hair follicles comes from the bottom-cell base [15], and the cause of the nude phenotype is a gene mutation of FoxN1, which leads to its loss of functionality [16]. FoxN1 mutations in humans cause immune deficiency, alopecia, nail dystrophia etc. Our results showed that the gene expression level of FoxN1 increased significantly on gestation day 95 (p<0.05). Previous research on the morphogenesis of secondary hair follicles of cashmere goats evidenced that the inner root sheath of the primary follicle, the outer root sheath, and the hair shaft developed until gestational day 95. At this time point of our study, a small amount of inner and outer root sheath could be observed from the side of secondary follicles, and the villi also began to form. On day 135, the development of the primary follicle was substantially complete, and part of the secondary hair follicles matured and the villi pierced the body surface, which indicated that FoxN1 may be involved in the differentiation of the inner root sheath, cortex, and medulla. The cyclical growth results showed that the expression of FoxN1 gene was overall elevated from April to October and reached a peak value in October that was significantly higher than those established at other stages (p<0.05). Further, from November to March, it declined continuously.

The expression trend of FoxN1 gene and the development cycle of hair follicles were basically consistent. Previous research on more mature follicles and growing hair follicles revealed that FoxN1 transcription occurred predominantly in the mature areas of the hair follicles, hair shaft, inner root sheath, and the outer root sheath [17]. Therefore, it could be speculated that FoxN1 plays an important role in the hair follicle growth. Reportedly, the mutations of FoxE1 gene caused Bamforth-Lazarus syndrome. The patients’ epiglottis was hypoplastic and bifid with sparse and spiky hairs, but the larynx was of normal diameter; the jaw was retrognathic [18]. Later, Clifton-Bligh et al [19] found that this was due to a mutation of codon 65, a substitution of alanine for valine. We established the expression level of FoxE1 from gestational day 95 to day 135 was relatively higher than those of the other periods. Moreover, its expression level showed an overall upward trend with the increase of gestational age, suggesting that FoxE1 gene is involved in hair follicle morphogenesis and plays a role in the development and maturation of hair follicles. It was expressed throughout the periodical growth process of the hair follicle, with a peak relative expression level in November. These findings are consistent with the reported conclusion that FoxE1 plays an important role in the embryonic development, cell growth, and differentiation. Therefore, it can be speculated that FoxE1 may be involved in the regulation process of periodical growth of the hair follicle.

Using the chip technology, researchers compared the DNA fragment of hairy and hairless dogs. They discovered that a mutation in the hairless dogs had been inherited from a unique ancestral, thus giving the now well-known name of the gene FoxI3. In dogs, FoxI3 haploinsufficiency leads to poor embryo development, which is characterized by an almost entire absence of hairs. In a mouse model, FoxI3 was found to show highly dynamic expression patterns in hair formation and cycle. In addition, the lack of FoxI3 prevents the downward growth of the hair follicles and hinders the progress of the hair cycle. Therefore, we can be certain FoxI3 regulates many aspects of hair follicle development and dynamic equilibrium. Vera Shirokova located the FoxI3 gene downstream of ectodysplasin (EDA) signaling pathways. And Noggin, BMP and EDA signaling molecules play an important role in the early developmental stages of follicle board. Thus, we further speculate FoxI3 gene may influence the origin and development of hair villi through interaction with other signaling molecules. Nevertheless, its role in the regulation of generation and development of the hair follicle needs further investigation. Our experiments demonstrated that the expression level of FoxI3 gene peaked on day 75, the level on day 65 was significantly higher than the expression level on day 45 (p<0.05). According to our results, the primary follicles in the Inner Mongolia cashmere goats begin to form from gestational day 55 to day 65, the secondary follicle growth occurs from day 65 to day 75, which indicates that FoxI3 gene may play a role in the secondary follicles initiation. FoxI3 was expressed in the skin tissue of adult cashmere goats all the year round, and its maximum relative expression level occurs in July. The results of our study show that the boundaries of each period are not absolute. The growth of the secondary follicles of Liaoning and Inner Mongolia cashmere goats was most active from July to November, with a peak in September. In November, the follicle cells began to die and catagen features appeared, and the processes in this month may be considered the transition from the growth phase to the catagen stage of secondary follicles. In January, follicles transit from catagen to their period of minimum activity – telogen, but in April, they transit back from this resting phase (telogen) to their growth phase (anagen). We hypothesize that FoxI3 may promote the growth of hair follicles in the anagen.

The production of goat cashmere and the quality of cashmere fibers are associated with the level of keratin. To study the important economic traits of cashmere sheep, the scholars at home and abroad mainly focus their investigations on the keratin gene family. Keratin is the most abundant protein in hair follicles and plays an important role in maintaining their structure. FoxN1 is a conservative transcription factor that regulates the expression of keratin in the skin. The absence or mutation of FoxN1 can cause hair shaft curl in the hair follicles due to cornification defects, which leads to the loss of its ability to penetrate the epidermis and form normal hair. The loss of the proteolytic activity of FoxN1 in humans and mice results in hypotrichosis, immunodeficiency (absence of thymus and T cells) and skin defects [20,21]. FoxN1 is expressed in the initial stage after the mitosis of hair follicle stromal cells. In the meantime, the stromal cells tend to differentiate in the direction of internal root sheath.

Based on the results of real-time qPCR of our examination reported here, we can conclude that the relative expression of the gene FoxN1 from gestation day 45 to gestation day 65 is relatively low since no expression of the respective protein was detected. The protein expression level exhibits an upward trend on gestation day 75. After day 75, the hair follicles grow and mature, and the structure of the hair shaft is gradually formed. Presumably, the protein FoxN1 can maintain the normal hair structure by regulating the expression of hair keratin. In the cyclical growth phase, the expression level of protein FoxN1 in September was higher than its levels in the other three periods. While the hair follicles entered the active period in September. In this period, the hair follicles of adult goats appear, develop, and rapidly produce a large amount of cashmere. Skin to express a large number of keratin and keratin associated protein. It is speculated that FoxN1 may play a catalytic role in the synthesis of keratin.

In previous studies, the expression of FoxE1 was found to be periodically suppressed in the quiescent stage, and then it induced the initiation of a new of growth period. The hair defects that can be observed are due to the abnormality in the hair follicle morphology, leading to the loss of the potential of hair follicles to penetrate correctly through the dermis and the subcutaneous tissue, which causes a deviation of the direction. Studies have found that protein FoxE1 is under expressed in the outer root sheath around the hair bulb and is a downstream target signal of Shh, participating in the later hair follicle morphogenesis. The lack of FoxE1 leads to abnormal extension of hair follicles to the depth of the dermis. The experimental results reported in this paper showed that the relative expression level of protein FoxE1 on gestation day 95 was higher than those in the other time points of gestational age. On gestation day 95, the stromal cells located at the end of the hair follicles surrounded the dermal papilla cells forming the hair bulbs. Meanwhile, the inner root sheaths and the outer root sheaths of some hair follicles could be observed. We speculate that the protein FoxE1 regulates the development of late hair follicle morphogenesis by interacting with a variety of signaling pathways. The experimental results reported in this paper showed that protein FoxE1 is expressed in March, June, September, and December. Presumably, FoxE1 may be involved in the cyclical growth of hair follicles. Its expression level in September is higher than the ones in the other months, because September is a relatively productive period in terms of hair follicle growth. Importantly, FoxE1 may be involved in the process of formation of the hair follicle.