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Simple Summary: Artificial breeding makes traits move forward in one direction and reach the extreme, such as ultra-fine wool covering the whole body of fine wool sheep. Nevertheless, many other domestic sheep remain the coarse wool type, and some mendelian genome loci have been identified as having major genes for these traits; however, the epigenetic regulation is still unclear. In our study, a set of lambs with coarse wool type all over their bodies were discovered within a full-sib family during an embryo transfer experiment of merino fine wool sheep. The difference between coarse and fine wool traits were studied from the perspective of RNA modification-N6-methyladenosine. A total of 31,153 peaks were collected, including 15,968 peaks in coarse skin samples and 15,185 peaks in fine skin samples. In addition, 7208 genes were differentially m6A methylated, including 4167 upregulated and 3041 downregulated in coarse skin samples. Four key genes (EDAR, FGF5, TCHH, KRT2) were obtained by comprehensive analysis of the MeRIP-seq and RNA sequence, which are closely related to primary wool follicle morphogenesis and development. The PI3K/AKT pathway was enriched through different m6A-related genes. These results provided new insights to understand the role of epigenetics in wool sheep domestication and breeding. [ABSTRACT FROM AUTHOR]

Abstract To investigate the optimal androgen concentration for culturing Hetian sheep wool follicle and to detect effects of androgen concentration on wool follicle cell proliferation and apoptosis using immunofluorescence labeling and real-time quantitative fluorescence determinations of wool keratin-associated protein gene expression levels. Wool follicles were isolated by microdissection and wool follicles and skin pieces were cultured in various concentrations of dihydrotestosterone (DHT) in culture medium. Next, daily lengthwise growth measurements of wool follicles were obtained using a microscopic micrometer. Cultured Hetian wool follicles were stained using the SACPIC method to reveal wool follicle structure, while sheep skin slices were used to observe cell proliferation by immunostaining and cell apoptosis using the TUNEL method. At the molecular biological level, keratin-associated protein (Kap) gene expression was studied using wool follicles cultured for various numbers of days in vitro. Effects of androgen concentrations on Hetian wool follicle growth and development were experimentally studied. EdU proliferation assays revealed that androgen promoted cell proliferation within wool follicle dermal papillae. TUNEL apoptosis detection demonstrated that androgen treatment could delay cell apoptosis. Quantitative reverse transcription polymerase chain reaction (qPCR) results demonstrated that gene expression level patterns of Hetian mountain sheep super-high sulfur protein. Kap1.1, KIF1.2, Kap2.12 and Kap4.2 gene expression level of the mountainous experimental group was significantly higher than plains Hetian sheep. An androgen concentration of 100 nM can promote the growth of Hetian wool follicle cells in vitro, resulting in overexpression of some genes of the Kap family.

Abstract Background Aohan fine wool sheep (AFWS) is a historically bred fine wool sheep, cultivated in China. The wool has excellent quality and good textile performance. Investigating the molecular mechanisms that regulate wool growth is important to improve wool quality and yield. Circular RNAs (circRNAs) are widely expressed non-coding RNAs that can act as competitive endogenous RNAs (ceRNAs) to bind to miRNAs. Although circRNAs have been studied in many fields, research on their activity in sheep wool follicles is limited. To understand the regulation of circRNAs in the growth of fine wool in sheep, we used RNA-Seq to identify circRNAs in sheep shoulder skin samples at three developmental stages: embryonic day 90 (E90d), embryonic day 120 (E120d), and at birth (Birth). Results We identified 8753 circRNAs and found that 918 were differentially-expressed. We then analyzed the classification and characteristic of the circRNAs in sheep shoulder skin. Using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), we identified the source genes of circRNAs, which were mainly enriched in cellular component organization, regulation of primary metabolic processes, tight junctions, and the cGMP-PKG and AMPK signaling pathways. In addition, we predicted interactions between 17 circRNAs and eight miRNAs, using miRanda software. Based on the significant pathways, we speculate that circ_0005720, circ_0001754, circ_0008036, circ_0004032, circ_0005174, circ_0005519, and circ_0007826 might play an important role in regulating wool follicle growth in AFWS. Seven circRNAs were randomly selected to validate the RNA-Seq results, using qRT-PCR. Conclusion Our results provide more information about circRNAs regulation of wool follicle development in AFWS, and establish a solid foundation for future research.

In our study, a set of lambs with coarse wool type all over their bodies were discovered within a full-sib family during an embryo transfer experiment of merino fine wool sheep. The difference between coarse and fine wool traits were studied from the perspective of RNA modification-N6-methyladenosine. A total of 31,153 peaks were collected, including 15,968 peaks in coarse skin samples and 15,185 peaks in fine skin samples. In addition, 7208 genes were differentially m6A methylated, including 4167 upregulated and 3041 downregulated in coarse skin samples. Four key genes (EDAR, FGF5, TCHH, KRT2) were obtained by comprehensive analysis of the MeRIP-seq and RNA sequence, which are closely related to primary wool follicle morphogenesis and development. The PI3K/AKT pathway was enriched through different m6A-related genes. These results provided new insights to understand the role of epigenetics in wool sheep domestication and breeding.

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Long non-coding RNAs (lncRNA) and microRNAs (miRNA) are new found classes of non-coding RNAs (ncRNAs) that are not translated into proteins but regulate various cellular and biological processes. In this study, we conducted a transcriptomic analysis of ncRNA and mRNA expression in Aohan fine wool sheep (AFWS) at different growth stages (embryonic day 90, embryonic day 120, and the day of birth), and explored their relationship with wool follicle growth. In total, 461 lncRNAs, 106 miRNAs, and 1,009 mRNAs were found to be differentially expressed during the three stages of wool follicle development. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to clarify the roles of the differentially expressed lncRNA, miRNA, and mRNA in the different stages of wool follicle development. Quantitative real-time PCR (qRT-PCR) was used to validate the results of RNA-seq analysis. lncRNA (MSTRG.223165) was found to act as a competing endogenous RNA (ceRNA) and may participate in wool follicle development by acting as an miR-21 sponge. Network prediction implicated the MSTRG.223165-miR-21-SOX6 axis in the wool follicle development. The targeting relationships of miR-21 with SOX6 and MSTRG.223165 were validated in dual-luciferase assays. This is the first report indicating the association of the lncRNA–miRNA–mRNA network with wool follicle development in AFWS. This study provides new insights into the regulation of the wool follicle growth and represents a solid foundation for wool sheep breeding programs.

Long non-coding RNAs (lncRNA) and microRNAs (miRNA) are new found classes of non-coding RNAs (ncRNAs) that are not translated into proteins but regulate various cellular and biological processes. In this study, we conducted a transcriptomic analysis of ncRNA and mRNA expression in Aohan fine wool sheep (AFWS) at different growth stages (embryonic day 90, embryonic day 120, and the day of birth), and explored their relationship with wool follicle growth. In total, 461 lncRNAs, 106 miRNAs, and 1,009 mRNAs were found to be differentially expressed during the three stages of wool follicle development. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to clarify the roles of the differentially expressed lncRNA, miRNA, and mRNA in the different stages of wool follicle development. Quantitative real-time PCR (qRT-PCR) was used to validate the results of RNA-seq analysis. lncRNA (MSTRG.223165) was found to act as a competing endogenous RNA (ceRNA) and may participate in wool follicle development by acting as an miR-21 sponge. Network prediction implicated the MSTRG.223165-miR-21- SOX6 axis in the wool follicle development. The targeting relationships of miR-21 with SOX6 and MSTRG.223165 were validated in dual-luciferase assays. This is the first report indicating the association of the lncRNA–miRNA–mRNA network with wool follicle development in AFWS. This study provides new insights into the regulation of the wool follicle growth and represents a solid foundation for wool sheep breeding programs. [ABSTRACT FROM AUTHOR]

Summary: Cryofixation by high‐pressure freezing (HPF) followed by freeze substitution (FS) is a preferred method to prepare biological specimens for ultrastructural studies. It has been shown to achieve uniform vitrification and ultrastructure preservation of complex structures in different cell types. One limitation of HPF is the small sample volume of <200 µm thickness and about 2000 µm across. A wool follicle is a rare intact organ in a single sample about 200 µm thick. Within each follicle, specialized cells derived from multiple cell lineages assemble, mature and cornify to make a wool fibre, which contains 95% keratin and associated proteins. In addition to their complex structure, large density changes occur during wool fibre development. Limited water movement and accessibility of fixatives are some issues that negatively affect the preservation of the follicle ultrastructure via conventional chemical processing. Here, we show that HPF‐FS of wool follicles can yield high‐quality tissue preservation for ultrastructural studies using transmission electron microscopy. [ABSTRACT FROM AUTHOR]

Background: Aohan fine wool sheep (AFWS) is a historically bred fine wool sheep, cultivated in China. The wool has excellent quality and good textile performance. Investigating the molecular mechanisms that regulate wool growth is important to improve wool quality and yield. Circular RNAs (circRNAs) are widely expressed non-coding RNAs that can act as competitive endogenous RNAs (ceRNAs) to bind to miRNAs. Although circRNAs have been studied in many fields, research on their activity in sheep wool follicles is limited. To understand the regulation of circRNAs in the growth of fine wool in sheep, we used RNA-Seq to identify circRNAs in sheep shoulder skin samples at three developmental stages: embryonic day 90 (E90d), embryonic day 120 (E120d), and at birth (Birth). Results: We identified 8753 circRNAs and found that 918 were differentially-expressed. We then analyzed the classification and characteristic of the circRNAs in sheep shoulder skin. Using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), we identified the source genes of circRNAs, which were mainly enriched in cellular component organization, regulation of primary metabolic processes, tight junctions, and the cGMP-PKG and AMPK signaling pathways. In addition, we predicted interactions between 17 circRNAs and eight miRNAs, using miRanda software. Based on the significant pathways, we speculate that circ_0005720, circ_0001754, circ_0008036, circ_0004032, circ_0005174, circ_0005519, and circ_0007826 might play an important role in regulating wool follicle growth in AFWS. Seven circRNAs were randomly selected to validate the RNA-Seq results, using qRT-PCR. Conclusion: Our results provide more information about circRNAs regulation of wool follicle development in AFWS, and establish a solid foundation for future research. [ABSTRACT FROM AUTHOR]

The apocrine sweat gland is a unique skin appendage in humans compared to mouse and chicken models. The absence of apocrine sweat glands in chicken and murine skin largely restrains further understanding of the complexity of human skin biology and skin diseases, like hircismus. Sheep may serve as an additional system for skin appendage investigation owing to the distributions and histological similarities between the apocrine sweat glands of sheep trunk skin and human armpit skin. To understand the molecular mechanisms underlying morphogenesis of apocrine sweat glands in sheepskin, transcriptome analyses were conducted to reveal 1631 differentially expressed genes that were mainly enriched in three functional groups (cellular component, molecular function and biological process), particularly in gland, epithelial, hair follicle and skin development. There were 7 Gene Ontology (GO) terms enriched in epithelial cell migration and morphogenesis of branching epithelium that were potentially correlated with the wool follicle peg elongation. An additional 5 GO terms were enriched in gland morphogenesis (20 genes), gland development (42 genes), salivary gland morphogenesis and development (8 genes), branching involved in salivary gland morphogenesis (6 genes) and mammary gland epithelial cell differentiation (4 genes). The enriched gland-related genes and two Kyoto Encyclopedia of Genes and Genomes pathway genes (WNT and TGF-β) were potentially involved in the induction of apocrine sweat glands. Genes named BMPR1A, BMP7, SMAD4, TGFB3, WIF1, and WNT10B were selected to validate transcript expression by qRT-PCR. Immunohistochemistry was performed to localize markers for hair follicle (SOX2), skin fibroblast (PDGFRB), stem cells (SOX9) and BMP signaling (SMAD5) in sheepskin. SOX2 and PDGFRB were absent in apocrine sweat glands. SOX9 and SMAD5 were both observed in precursor cells of apocrine sweat glands and later in gland ducts. These results combined with the upregulation of BMP signaling genes indicate that apocrine sweat glands were originated from outer root sheath of primary wool follicle and positively regulated by BMP signaling. This report established the primary network regulating early development of apocrine sweat glands in sheepskin and will facilitate the further understanding of histology and pathology of apocrine sweat glands in human and companion animal skin.

The apocrine sweat gland is a unique skin appendage in humans compared to mouse and chicken models. The absence of apocrine sweat glands in chicken and murine skin largely restrains further understanding of the complexity of human skin biology and skin diseases, like hircismus. Sheep may serve as an additional system for skin appendage investigation owing to the distributions and histological similarities between the apocrine sweat glands of sheep trunk skin and human armpit skin. To understand the molecular mechanisms underlying morphogenesis of apocrine sweat glands in sheepskin, transcriptome analyses were conducted to reveal 1631 differentially expressed genes that were mainly enriched in three functional groups (cellular component, molecular function and biological process), particularly in gland, epithelial, hair follicle and skin development. There were 7 Gene Ontology (GO) terms enriched in epithelial cell migration and morphogenesis of branching epithelium that were potentially correlated with the wool follicle peg elongation. An additional 5 GO terms were enriched in gland morphogenesis (20 genes), gland development (42 genes), salivary gland morphogenesis and development (8 genes), branching involved in salivary gland morphogenesis (6 genes) and mammary gland epithelial cell differentiation (4 genes). The enriched gland-related genes and two Kyoto Encyclopedia of Genes and Genomes pathway genes (WNT and TGF-β) were potentially involved in the induction of apocrine sweat glands. Genes named BMPR1A, BMP7, SMAD4, TGFB3, WIF1 , and WNT10B were selected to validate transcript expression by qRT-PCR. Immunohistochemistry was performed to localize markers for hair follicle (SOX2), skin fibroblast (PDGFRB), stem cells (SOX9) and BMP signaling (SMAD5) in sheepskin. SOX2 and PDGFRB were absent in apocrine sweat glands. SOX9 and SMAD5 were both observed in precursor cells of apocrine sweat glands and later in gland ducts. These results combined with the upregulation of BMP signaling genes indicate that apocrine sweat glands were originated from outer root sheath of primary wool follicle and positively regulated by BMP signaling. This report established the primary network regulating early development of apocrine sweat glands in sheepskin and will facilitate the further understanding of histology and pathology of apocrine sweat glands in human and companion animal skin. [ABSTRACT FROM AUTHOR]

The high concentration of secondary branched wool follicles is a distinctive feature of the Merino sheep. At present, the molecular control of the development and branching of secondary wool follicles (SF) remains elusive. To reveal the potential genes associated with the development of hair follicles, we investigated the characteristics of prenatal and postnatal development of wool follicles, and the transcriptional expression profile in fetuses/lambs from dams under either maternal maintenance or sub-maintenance (75% maintenance) nutrition. The density of SF and the ratio of SF to primary wool follicles (PF) were reduced (p < 0.05) in fetuses from day 105 to 135 of gestation under sub-maintenance nutrition. Differentially expressed genes were enriched in the binding, single-organism process, cellular process, cell and cell part Gene Ontology (GO) functional categories and metabolism, apoptosis, and ribosome pathways. Four candidate genes, SFRP4, PITX1, BAMBI, and KRT16, which were involved in secondary wool follicles branching and development, were identified. Our results indicate that nutritional intervention imposed on pregnant ewes by short-term sub-maintenance nutrition could provide a strategy for the study of wool follicle development. Overall insight into the global gene expression associated with SF development can be used to investigate the underlying mechanisms of SF branching in Merino sheep.

Murine primary hair follicle induction is driven by the communication between the mesenchyme and epithelium and mostly governed by signaling pathways including wingless-related integration site (WNT), ectodysplasin A receptor (EDAR), bone morphogenetic protein (BMP), and fibroblast growth factor (FGF), as observed in genetically modified mouse models. Sheep skin may serve as a valuable system for hair research owing to the co-existence of sweat glands with wool follicles in trunk skin and asynchronized wool follicle growth pattern similar to that of human head hair follicles. However, the mechanisms underlying wool follicle development remain largely unknown. To understand how long non-coding RNAs (lncRNAs) and mRNAs function in primary wool follicle induction in carpet wool sheep, we conducted high-throughput RNA sequencing and revealed globally altered lncRNAs (36 upregulated and 26 downregulated), mRNAs (228 elevated and 225 decreased), and 80 differentially expressed novel transcripts. Several key signals in WNT (WNT2B and WNT16), BMP (BMP3, BMP4, and BMP7), EDAR (EDAR and EDARADD), and FGF (FGFR2 and FGF20) pathways, and a series of lncRNAs, including XLOC_539599, XLOC_556463, XLOC_015081, XLOC_1285606, XLOC_297809, and XLOC_764219, were shown to be potentially important for primary wool follicle induction. GO and KEGG analyses of differentially expressed mRNAs and potential targets of altered lncRNAs were both significantly enriched in morphogenesis biological processes and transforming growth factor-β, Hedgehog, and PI3K-Akt signaling, as well as focal adhesion and extracellular matrix-receptor interactions. The prediction of mRNA-mRNA and lncRNA-mRNA interaction networks further revealed transcripts potentially involved in primary wool follicle induction. The expression patterns of mRNAs and lncRNAs of interest were validated by qRT-PCR. The localization of XLOC_297809 and XLOC_764219 both in placodes and dermal condensations was detected by in situ hybridization, indicating important roles of lncRNAs in primary wool follicle induction and skin development. This is the first report elucidating the gene network of lncRNAs and mRNAs associated with primary wool follicle early development in carpet wool sheep and will shed new light on selective wool sheep breeding.

β-Catenin is an evolutionarily conserved molecule in the canonical Wnt signaling pathway, which controls decisive steps in embryogenesis and functions as a crucial effector in the development of hair follicles. However, the molecular mechanisms underlying wool production have not been fully elucidated. In this study, we investigated the effects of ovine β-catenin on wool follicles of transgenic sheep produced by pronuclear microinjection with a skin-specific promoter of human keratin14 (k14). Both polymerase chain reaction and Southern blot analysis showed that the sheep carried the ovine β-catenin gene and that the β-catenin gene could be stably inherited. To study the molecular responses to high expression of β-catenin, high-throughput RNA-seq technology was employed using three transgenic sheep and their wild-type siblings. These findings suggest that β-catenin normally plays an important role in wool follicle development by activating the downstream genes of the Wnt pathway and enhancing the expression of keratin protein genes and keratin-associated protein genes.

Thyroidectomy surgery performed late in gestation results in perturbations in wool follicle development in foetal sheep, showing the importance of thyroid hormones for wool follicle development. The aim of this study was to determine the influence of transient manipulation of thyroid hormone status at a time corresponding with foetal primary wool follicle initiation. Pregnant Merino ewes (n = 12 per treatment) were treated daily between gestational days 55 and 64 with control (vehicle), exogenous thyroxine (T4) or propylthiouracil (PTU), an inhibitor of T4 synthesis, and conversion to the active form of the thyroid hormone (triiodothyronine). There were no significant differences in birth weight, gestational lengths and birth coat scores of the resultant lambs. The total primary and secondary follicle densities were significantly lower in lambs exposed to exogenous T4 compared with other treatments (P < 0.05). However, the T4 group displayed a higher proportion of mature secondary follicles (reflected by increased mature secondary follicle densities and mature secondary/primary follicle ratios) than the other treatment groups (P < 0.05). The skin morphology of the lambs differed 12 months later, with the T4 group having significantly higher total follicle densities compared with the PTU group, largely attributed to increased mature and total secondary follicle densities. However, this increase in wool follicle densities did not translate to differences in the fleece yields and weight, fibre diameter, staple lengths or any other fibre parameters. This study showed that transient manipulation of thyroid hormone status during foetal primary follicle initiation does have long-term consequences on the morphology of wool follicles, in particular the maturity of secondary wool follicles.

HOXC-13 has an important role in controlling hair formation through regulating keratin differentiation-specific genes. In this study, we describe the isolation and characterisation of the Hoxc-13 gene from sheep wool follicles and its expression in the skin. We show that the gene organisation of ovine Hoxc-13 is similar to other homeobox genes of the Abd-B type I Homeobox class with two exons split by an intron next to the homeobox. The gene spans 7.5 kilobases (kb) and has a relatively large intron, which divides an open reading frame of 2361 nucleotides. The predicted ovine Hoxc-13 protein of 330 amino acids has over 97% sequence identity with the human and mouse proteins. A second novel transcript was identified, which could produce a truncated Hoxc-13 protein lacking 15 amino acids from the N-terminus. A positionally conserved Hoxc-13 binding site in the Hoxc-13 proximal promoters of sheep, human, mouse and newt suggests that Hoxc-13 expression is autoregulatory. Positionally conserved motifs for LEF-1 and Whn/Foxn1 suggest that Hoxc-13 may be a downstream target of these transcription factors known to regulate hair growth. In addition to expression in the follicle, we detected Hoxc-13 in cells of the blood sinus surrounding vibrissal follicles and in scattered cells in the upper dermis of the skin. Thus, in addition to a role in controlling transcription of hair keratins, Hoxc-13 may have other roles in skin function. [Copyright &y& Elsevier]

We sequenced 2939 ESTs from fetal and adult sheep skin. Stages of gestation were picked to coincide with the major events in skin appendage (wool follicle) formation. Clustering analysis generated a nonredundant set of ESTs 2435 strong (83% nonredundant). Approximately 24% of these gave no hit to NCBI build 29 of the human genome, while 35% were tentatively classified by putative function based on BLASTX hits with a p(N) of <10−4. In addition to bioinformatics analysis of our ESTs and gene mapping, we have generated a large EST spatial expression data set using in situ hybridization. One thousand one hundred forty-two ESTs have been used for in situ localization; about 31% are from adult sheep skin, 39% from late gestation fetal sheep skin, and 30% from midgestation fetal sheep skin. These probes have been used in over 3000 hybridization experiments. In this report, we summarize the results of in situs on adult sheep skin. [Copyright &y& Elsevier]

In the hair follicle the cuticle develops as a thin layer of cells between the hair shaft cortex and the inner root sheath. Once the cuticle cells begin to differentiate they accumulate cysteine-rich granules in their cytoplasm but the identity of their constituent proteins has remained largely an enigma. In this report we show differential expression of a family of genes encoding cysteine-rich, glycine-rich keratins in the cuticle. Two clones of the sheep KAP5 gene family were isolated: the KAP5.4 cDNA encodes a protein of 190 amino acids (Mr = 16,936) containing 32 mol% cysteine, 26 mol% glycine and the partial KAP5.5 cDNA encodes a protein of at least 197 amino acids (Mr ≥ 17,474) containing 29 mol% cysteine, 28 mol% glycine. The predicted amino acid sequences of the KAP5 family show extensive sequence conservation and all the proteins are composed almost entirely of cysteine-rich and glycine-rich repeats. Each KAP5 gene produces an ∼ 1.5- kb mRNA species but the KAP5.4 and KAP5.5 mRNA levels appear to be severalfold greater than the KAP5.1 mRNA. Comparative tissue in situ hybridizations reveal a positive correlation between the onset of expression and follicle depth. For a given KAP5 gene two widely different cuticle expression patterns were noted amongst the follicle populations, and on the basis of follicle bulb cell kinetics they are consistent with expression in either sheep primary or secondary follicle types. [ABSTRACT FROM AUTHOR]

A procedure for the culture of isolated wool follicles from Merino sheep is described. Follicles were microdissected from midside skin samples of 2-yr-old wethers and transferred, individually, to 24-well tissue culture plates. When maintained in supplemented Williams' E medium containing 5 to 10% fetal bovine serum (FBS), insulin, hydrocortisone, and a trace element mixture, fiber growth rates of 40 to 80 µm/day were observed. Follicles maintained their morphologic integrity for up to 7 days, incorporated [methyl-H]thymidine into DNA and [S]methionine into intermediate-filament keratins of the growing fiber. Insulin and hydrocortisone stimulated fiber growth at concentrations of 10 µg/ml and 50 ng/ml, respectively, but higher doses were inhibitory. The growth of fibers in response to hydrocortisone and the changes in follicle morphology was similar to those induced in skin after systemic administration of cortisol in vivo. A positive interaction between hydrocortisone and trace elements for follicle survival and hydrocortisone, insulin, and FBS for fiber growth was also found. The successful culture of Merino sheep follicles provides a model with which to study the direct influence of endocrine, nutritional and local factors on wool keratin synthesis independently of systemic shifts in the animals' metabolism. [ABSTRACT FROM AUTHOR]

• Skin sub-regions are different in morphology between coarse and fine wool sheep. • Molecular variations drive delicate skin differences between sheep lines. • Candidate genes are correlated with dermal condensate and dermal fibroblast. • APCDD1, IGFBP3 and SFRP4 etc. are potential effectors regulating skin compartments. The hair follicle is an excellent mini-system illustrating the mechanisms governing organogenesis and regeneration. Although the general mechanisms modulating skin and hair follicle development are widely studied in mouse and chicken models, the delicate network regulating skin and hair diversity remains largely unclear. Sheep is an additional model to address the various wool characteristics observed in nature. The coarse and fine wool sheep with diverse fibers were examined to show differences in the primary wool follicle size and skin thickness. The molecular dynamics in skin staged at the primary wool follicle induction between two sheep lines were investigated by RNA-sequencing analyses to generate 1994 differentially expressed genes revealing marker genes for epithelium (6 genes), dermal condensate (38 genes) and dermal fibroblast (58 genes) highly correlated with skin and wool follicle morphological differences. The DEGs were enriched in GO terms represented by epithelial cell migration and differentiation, regulation of hair follicle development and ectodermal placode formation, and KEGG pathways typified by WNT and Hedgehog signaling pathways governing the differences of skin structure. The qPCR detection of 9 genes confirmed the similar expression tendency with RNA-sequencing profiles. This comparative study of coarse and fine wool sheep skin reveals the presence of skin and wool follicle differences at primary wool follicle induction stage, and indicates the potential effectors (APCDD1 , FGF20, DKK1 , IGFBP3 and SFRP4) regulating the skin compartments during the early morphogenesis of primary wool follicles to shape the variable wool fiber thickness in later developmental stages. [ABSTRACT FROM AUTHOR]

Simple Summary: The morphogenesis hair follicles begins in the embryonic stage. The first follicles formed are the primary wool follicles (PF), followed by secondary wool follicles (SF), and then secondary-derived follicles that branch from the SF in Merino sheep. The morphogenesis and development of SF in sheep determine wool yield and quality. At present, the mechanism of SF development and branching is still unclear. In this study, hair follicle morphogenesis and development were inhibited through nutritional restrictions during pregnancy, and transcriptome sequencing was performed to screen for differentially expressed genes related to the SF development in the fetus. Our findings are helpful to understand the mechanisms of SF development and branching of Merino sheep. The high concentration of secondary branched wool follicles is a distinctive feature of the Merino sheep. At present, the molecular control of the development and branching of secondary wool follicles (SF) remains elusive. To reveal the potential genes associated with the development of hair follicles, we investigated the characteristics of prenatal and postnatal development of wool follicles, and the transcriptional expression profile in fetuses/lambs from dams under either maternal maintenance or sub-maintenance (75% maintenance) nutrition. The density of SF and the ratio of SF to primary wool follicles (PF) were reduced (p < 0.05) in fetuses from day 105 to 135 of gestation under sub-maintenance nutrition. Differentially expressed genes were enriched in the binding, single-organism process, cellular process, cell and cell part Gene Ontology (GO) functional categories and metabolism, apoptosis, and ribosome pathways. Four candidate genes, SFRP4, PITX1, BAMBI, and KRT16, which were involved in secondary wool follicles branching and development, were identified. Our results indicate that nutritional intervention imposed on pregnant ewes by short-term sub-maintenance nutrition could provide a strategy for the study of wool follicle development. Overall insight into the global gene expression associated with SF development can be used to investigate the underlying mechanisms of SF branching in Merino sheep. [ABSTRACT FROM AUTHOR]

β-Catenin is an evolutionarily conserved molecule in the canonical Wnt signaling pathway, which controls decisive steps in embryogenesis and functions as a crucial effector in the development of hair follicles. However, the molecular mechanisms underlying wool production have not been fully elucidated. In this study, we investigated the effects of ovine β-catenin on wool follicles of transgenic sheep produced by pronuclear microinjection with a skin-specific promoter of human keratin14 (k14). Both polymerase chain reaction and Southern blot analysis showed that the sheep carried the ovine β-catenin gene and that the β-catenin gene could be stably inherited. To study the molecular responses to high expression of β-catenin, high-throughput RNA-seq technology was employed using three transgenic sheep and their wild-type siblings. These findings suggest that β-catenin normally plays an important role in wool follicle development by activating the downstream genes of the Wnt pathway and enhancing the expression of keratin protein genes and keratin-associated protein genes. [ABSTRACT FROM AUTHOR]

Murine primary hair follicle induction is driven by the communication between the mesenchyme and epithelium and mostly governed by signaling pathways including wingless-related integration site (WNT), ectodysplasin A receptor (EDAR), bone morphogenetic protein (BMP), and fibroblast growth factor (FGF), as observed in genetically modified mouse models. Sheep skin may serve as a valuable system for hair research owing to the co-existence of sweat glands with wool follicles in trunk skin and asynchronized wool follicle growth pattern similar to that of human head hair follicles. However, the mechanisms underlying wool follicle development remain largely unknown. To understand how long non-coding RNAs (lncRNAs) and mRNAs function in primary wool follicle induction in carpet wool sheep, we conducted high-throughput RNA sequencing and revealed globally altered lncRNAs (36 upregulated and 26 downregulated), mRNAs (228 elevated and 225 decreased), and 80 differentially expressed novel transcripts. Several key signals in WNT ( WNT2B and WNT16 ), BMP ( BMP3, BMP4 , and BMP7 ), EDAR ( EDAR and EDARADD ), and FGF ( FGFR2 and FGF20 ) pathways, and a series of lncRNAs, including XLOC&#95;539599, XLOC&#95;556463, XLOC&#95;015081, XLOC&#95;1285606, XLOC&#95;297809, and XLOC&#95;764219, were shown to be potentially important for primary wool follicle induction. GO and KEGG analyses of differentially expressed mRNAs and potential targets of altered lncRNAs were both significantly enriched in morphogenesis biological processes and transforming growth factor-β, Hedgehog, and PI3K-Akt signaling, as well as focal adhesion and extracellular matrix-receptor interactions. The prediction of mRNA-mRNA and lncRNA-mRNA interaction networks further revealed transcripts potentially involved in primary wool follicle induction. The expression patterns of mRNAs and lncRNAs of interest were validated by qRT-PCR. The localization of XLOC&#95;297809 and XLOC&#95;764219 both in placodes and dermal condensations was detected by in situ hybridization, indicating important roles of lncRNAs in primary wool follicle induction and skin development. This is the first report elucidating the gene network of lncRNAs and mRNAs associated with primary wool follicle early development in carpet wool sheep and will shed new light on selective wool sheep breeding.