Characterization and functional analysis of lncRNA2690 in regulating the growth cycle of the hair follicle in rabbits.

Hair follicles (HFs) achieve hair growth and renewal by periodic regeneration. Therefore, exploring the key factors affecting hair growth in rabbits is of great significance for precisely breeding Angora rabbits and improving the competitiveness of the rabbit industry. Based on the results of our previous studies, lncRNA2690 was differentially expressed in the HF cycle using lncRNA-Seq, and the full-length sequence was annotated by bioinformatics analysis. The lncRNA2690 is 363 nt long and is found on chromosome 14 from 163 321 514 to 163 321 872. The lncRNA2690 was predicted to not have the coding ability through open reading frame and CPC2, and the nuclear-cytoplasmic separation experiment showed the lncRNA2690 to be highly expressed in the nucleus (p < 0.01). The expression pattern of lncRNA2690 was further analyzed in the different HF development stages of Angora rabbits using quantitative real-time PCR. The results showed that lncRNA2690 was periodically expressed in HF development, and the expression level was found to be high in the HF resting phases. The overexpression and knockdown of lncRNA2690 were found to significantly upregulate and downregulate the expression of the genes WNT2, CCND1, BMP2, LEF1, and SIAH1 in the rabbit dermal papilla cells (p < 0.01), promoting cell apoptosis and inhibiting cell proliferation (p < 0.01). This indicated that lncRNA2690 negatively regulates the periodic regeneration of the HFs in rabbits. These results provide a basis for the further study of lncRNA2690 in the HF growth cycle of Angora rabbits.

Establishment of Angora rabbits' whisker hair follicle model and optimal culture conditions.

To establish the model of whisker hair follicle culture in vitro and explore the best culture conditions, the whisker hair follicles of Angora rabbits were separated with stereomicroscope and cultured in William's E, DMEM, MEM media. The surface of the cultured whisker hair follicles was not damaged due to manual operation, resulting in the hair shaft's growth. This indicated the success of the in vitro whisker hair follicle model. The hair shaft grew at the fastest rate in the William's E culture (p < 0.05), which was significantly higher than that in the DMEM and MEM media. The hematoxylin-eosin results showed that compared to the William's E group, the atrophy of whisker hair follicles in the DMEM and MEM media was evident, especially in the MEM medium. PCNA immunofluorescence staining was employed to detect the expression of whisker hair follicles. The results showed that the PCNA positive expression of the William's E group was significantly stronger than that of the DMEM and MEM groups. Furthermore, CCK-8 and Annexin V-FITC/PI methods were used to detect the proliferation and apoptosis of the dermal papilla cells (DPCs). The results of this study provide a model for studying the hair growth of fur animals.

Analysis of histology and long noncoding RNAs involved in the rabbit hair follicle density using RNA sequencing.

BACKGROUND: Hair follicle density influences wool fibre production, which is one of the most important traits of the Wan Strain Angora rabbit. However, molecular mechanisms regulating hair follicle density have remained elusive. RESULTS: In this study, hair follicle density at different body sites of Wan Strain Angora rabbits with high and low wool production (HWP and LWP) was investigated by histological analysis. Haematoxylin-eosin staining showed a higher hair follicle density in the skin of the HWP rabbits. The long noncoding RNA (lncRNA) profile was investigated by RNA sequencing, and 50 and 38 differentially expressed (DE) lncRNAs and genes, respectively, were screened between the HWP and LWP groups. A gene ontology analysis revealed that phospholipid, lipid metabolic, apoptotic, lipid biosynthetic, and lipid and fatty acid transport processes were significantly enriched. Potential functional lncRNAs that regulate lipid metabolism, amino acid synthesis, as well as the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) and hedgehog signalling pathways, were identified. Consequently, five lncRNAs (LNC_002171, LNC_000797, LNC_005567, LNC_013595, and LNC_020367) were considered to be potential regulators of hair follicle density and development. Three DE lncRNAs and genes were validated by quantitative real-time polymerase chain reaction (q-PCR). CONCLUSIONS: LncRNA profiles provide information on lncRNA expression to improve the understanding of molecular mechanisms involved in the regulation of hair follicle density.

DNA methylation and histone acetylation are involved in Wnt10b expression during the secondary hair follicle cycle in Angora rabbits.

Secondary hair follicles (SHFs) in the Angora rabbit exhibit classic cyclic hair development, but the multiple molecular signals involved in hair cycling are yet to be explored in detail. In the present study, we investigated the expression pattern, methylation and histone H3 acetylation status of Wnt10b, as a molecular signal participating in hair cycling, during the SHF cycle in the Angora rabbit. Expression of Wnt10b at the anagen phase was significantly higher than that at both the telogen and catagen phases, suggesting that Wnt10b might serve as a critical activator during cyclic transition of SHFs. Methylation frequency of the fifth CpG site (CpG5-175 bp) in CpG islands at the anagen phase was lower than that at both the catagen and telogen phases. The methylation status of the CpG5 site was negatively correlated with Wnt10b expression. This indicated that the methylation of CpG5 might participate in Wnt10b transcriptional suppression in SHFs. Furthermore, histone H3 acetylation status in the regions-256~-11 bp and 98 ~ 361 bp were significantly lower at both the catagen and telogen phases than at the anagen phase. The histone H3 acetylation level was significantly positively correlated with Wnt10b expression. This confirmed that histone acetylation was likely involved in upregulating Wnt10b transcription in SHFs. Additionally, potential binding to the transcription factors ZF57 and HDBP was predicted within the CpG5 site. In conclusion, our findings reveal the epigenetic mechanism of Wnt10b transcription and provide a new insight into epigenetic regulation during the SHF cycle in the Angora rabbit.

Characterization of HTATIP2 and its role during hair follicle cycles in Angora rabbit.

Hair follicle (HF) growth and cycling is a complex biological process that occurs in most mammals. As HF growth and cycling directly impacts rabbit wool yield, it is important to better understand the potential regulation pattern of HF development. Our previous study demonstrated that HTATIP2 may participate in regulating rabbit HF cycles, but the molecular mechanism of HTATIP2 remained unclear. In this study, the coding sequence of the HTATIP2 gene in Angora rabbit was cloned. The length of the coding region sequence was 840 bp, which could code 279 amino acids, and exhibited high homology in different mammals. Bioinformatics analyses indicated that the HTATIP2 protein is stable, hydrophilic, located around the cytoplasm, and has a putative signal peptide. Moreover, we verified that HTATIP2 is highly expressed during catagen and telogen of the HF cycle. The overexpression vector was constructed and siRNAs were designed. Overexpression and knockdown of HTATIP2 appeared to regulate JAK-STAT pathway genes, such as BCL2, CCND1, c-Myc, and STAT2. It is therefore likely that HTATIP2 promotes cell apoptosis and inhibits cell proliferation. Our results indicate that HTATIP2 is highly expressed during catagen and telogen and may play an important role in JAK-STAT signaling. This study provides a theoretical foundation for investigating HTATIP2 in biological processes.

Characterization and functional analysis of SIAH1 during skin and hair follicle development in the angora rabbit (Oryctolagus cuniculus).

BACKGROUND: Seven in absentia homolog 1 (SIAH1) is an E3 ubiquitin ligase containing a RING-finger domain and a key regulator of normal development. Skin and hair follicle development is a complex and special process of morphogenesis involving multiple signaling pathways. SIAH1 is enriched in the Wnt signaling pathway and potentially related to hair follicle cycle and skin development. This study aims to provide evidence for the role of SIAH1 in skin and hair development. RESULTS: Full-length cloning and analysis of SIAH1 was conducted to better understand its function. Phylogenetically, the sequence of SIAH1 in the rabbit shares the greatest homology with Home sapiens, Pongo abelii and Mus mulatta. Based on the rabbit hair follicle synchronization model, we found that the expression level of SIAH1 in the regressive period of the rabbit hair cycle is significantly lower than in the active growth and rest periods. In addition, the mRNA expression levels of skin and hair follicle development-related genes changed significantly when SIAH1 was overexpressed and silenced. After SIAH1 overexpression, the expression levels of WNT2, LEF1 and FGF2 decreased, and those of SFRP2 and DKK1 increased (P < 0.05). After interference of SIAH1, the expression levels of WNT2, LEF1 and FGF2 increased (P < 0.05), and SFRP2 and DKK1 decreased. CONCLUSIONS: SIAH1 can affect skin and hair follicle development and exert an inhibitory effect. These results could provide foundamental insights into the role of SIAH1 as a target gene in rabbit skin and hair follicle development.

Effects of the dietary digestible fiber-to-starch ratio on pellet quality, growth and cecal microbiota of Angora rabbits.

OBJECTIVE: Substituting starch with digestible fiber (dF) can improve digestive health of rabbits and reduce costs. Therefore, it is necessary to develop a criterion for dF and starch supply. Effects of the dietary dF-to-starch ratio on pellet quality, growth and cecal microbiota of Angora rabbits were evaluated. METHODS: Five isoenergetic and isoproteic diets with increasing dF/starch ratios (0.59, 0.66, 0.71, 1.05, and 1.44) were formulated. A total of 120 Angora rabbits with an average live weight of 2.19 kg were randomly divided into five groups with four replicates. At the end of 40 day feeding trial, cecal digesta were collected to analyse microbiota. RESULTS: The results showed that the dF/starch ratio had linear effects on pellet variables (p<0.01). When the dF/starch ratio was 1.44, the pellets had the lowest powder and highest durability. The dF/starch ratio had unfavorable linear effects on growth variables (p<0.001). When analyzed by quadratic regression, the optimal dF/starch ratios for average weight gain and feed/gain were 0.59 and 0.74, respectively. There were differences in wool yield, fiber length and fiber diameter caused by the dF/starch ratio (p<0.05), and the dF/starch ratios that ranged from 0.66 to 1.06 were appropriate for good results. The cecal microbiota operational taxonomic unit (OTU) number index in the 1.05 dF/starch treatment was higher than that in the 0.66 and 0.71 dF/starch treatments. The higher dF/starch ratio resulted in a higher cecal microbiota OTU number index (p<0.05). The proportion of Ruminococcus in the 0.71 dF/starch treatment was higher than that in the 0.59 dF/starch treatment (p<0.05). CONCLUSION: The most suitable dF/starch ratio for feed pellet quality is 1.44, and for rabbit growth the optimal range of ratios is from 0.59 to 0.74. With combination of the wool growth, output cost, and cecal microbiota, we suggest that a dietary dF/starch ratio ranging from 0.74 to 1.06 is optimal.

Effects of Wnt10b on dermal papilla cells via the canonical Wnt/ß-catenin signalling pathway in the Angora rabbit.

Wnt10b is a member of Wnt family that plays a variety of roles in biological functions, including those in the development of hair follicles. To investigate the effect of Wnt10b on hair growth in the Angora rabbit and to determine the underlying molecular mechanism, we cultured dermal papilla (DP) cells with exogenous Wnt10b in vitro. We observed the expressions of downstream critical gene ß-catenin and lymphoid enhancer-binding factor 1 (LEF1) in Wnt/ß-catenin pathway. The levels of ß-catenin mRNA and protein were higher in the Wnt10b group of DP cells than in the Control group, and the mRNA level of LEF1 in the Wnt10b group was higher than in the Control group. Moreover, translocation of ß-catenin from cytoplasm to nucleus was activated in the Wnt10b group. Furthermore, the mRNA levels of the hair follicle-regulatory genes, insulin-like growth factor-1 (IGF-1) and alkaline phosphatase (ALP), and the protein activity of ALP was also upregulated in the Wnt10b group compared to their corresponding levels in the Control group. These data suggest that Wnt10b could activate the canonical Wnt/ß-catenin signalling pathway to induce DP cells in the Angora rabbit. In addition, the proliferation of DP cells was significantly promoted when cultured with Wnt10b for 48 and 72 hr, suggesting that Wnt10b plays a pivotal role in the proliferation and maintenance of DP cells in vitro. In conclusion, this study demonstrates that Wnt10b may promote hair follicle growth in Angora rabbit through the canonical Wnt/ß-catenin signalling pathway that promotes the proliferation of DP cells.

Intestinal macrophages in Peyer's patches, sacculus rotundus and appendix of Angora rabbit.

The largest pool of macrophages in the body is harboured by the intestinal mucosa. As the principal phagocytic component of the immune system, macrophages are essential for maintaining mucosal homeostasis as they prevent commensal bacteria from adhering to mucosal epithelial cells. This study provides a RAM11 immunohistochemical and electron microscopic investigation of the existence, localization and distribution of intestinal macrophages in organized gut-associated lymphoid tissue (GALT), including Peyer's patches (PPs), the sacculus rotundus (SR) and the appendix, in the Angora rabbit. Although rabbit intestinal macrophages did not express the tissue macrophage marker macrosialin (CD68), they expressed RAM11. RAM11-positive intestinal macrophages were mostly localized to the subepithelial dome region, interfollicular area and germinal centres (GCs) of the GALT and the lamina propria or submucosa of the ileum and jejunum devoid of PPs and were also observed in the follicle-associated epithelium of PPs, but not in that of the SR and appendix. RAM11-positive macrophages containing engulfed apoptotic bodies were present in the GCs of the lymphoid follicles in the GALT. Electron microscopy further revealed multiple macrophages containing apoptotic bodies within the GCs of the follicles in the GALT. Some macrophage aggregations were observed in the GC and between the GC and the corona region of the follicles in the SR and appendix. Rabbit intestinal macrophages thus undertake both potent phagocytic activity and the efficient scavenging of apoptotic cells. Immunohistochemical data suggest that RAM11 can be reliably used for the determination of intestinal macrophages in the GALT of rabbits.

Regulation of Hair Follicle Growth and Development by Different Alternative Spliceosomes of FGF5 in Rabbits.

This study investigated the regulatory effect of alternative spliceosomes of the fibroblast growth factor 5 (FGF5) gene on hair follicle (HF) growth and development in rabbits. The FGF5 alternative spliceosomes (called FGF5-X1, FGF5-X2, FGF5-X3) were cloned. The overexpression vector and siRNA of spliceosomes were transfected into dermal papilla cells (DPCs) to analyze the regulatory effect on DPCs. The results revealed that FGF5-X2 and FGF5-X3 overexpression significantly decreased LEF1 mRNA expression (p < 0.01). FGF5-X1 overexpression significantly reduced CCND1 expression (p < 0.01). FGF5-X1 and FGF5-X2 possibly downregulated the expression level of FGF2 mRNA (p < 0.05), and FGF5-X3 significantly downregulated the expression level of FGF2 mRNA (p < 0.01). The FGF5 alternative spliceosomes significantly downregulated the BCL2 mRNA expression level in both cases (p < 0.01). FGF5-X1 and FGF5-X2 significantly increased TGFß mRNA expression (p < 0.01). All three FGF5 alternative spliceosomes inhibited DPC proliferation. In conclusion, the expression profile of HF growth and development-related genes can be regulated by FGF5 alternative spliceosomes, inhibiting the proliferation of DPCs and has an influence on the regulation of HF growth in rabbits. This study provides insights to further investigate the mechanism of HF development in rabbits via FGF5 regulation.

Marker density and statistical model designs to increase accuracy of genomic selection for wool traits in Angora rabbits.

The Angora rabbit, a well-known breed for fiber production, has been undergoing traditional breeding programs relying mainly on phenotypes. Genomic selection (GS) uses genomic information and promises to accelerate genetic gain. Practically, to implement GS in Angora rabbit breeding, it is necessary to evaluate different marker densities and GS models to develop suitable strategies for an optimized breeding pipeline. Considering a lack in microarray, low-coverage sequencing combined with genotype imputation was used to boost the number of SNPs across the rabbit genome. Here, in a population of 629 Angora rabbits, a total of 18,577,154 high-quality SNPs were imputed (imputation accuracy above 98%) based on low-coverage sequencing of 3.84X genomic coverage, and wool traits and body weight were measured at 70, 140 and 210 days of age. From the original markers, 0.5K, 1K, 3K, 5K, 10K, 50K, 100K, 500K, 1M and 2M were randomly selected and evaluated, resulting in 50K markers as the baseline for the heritability estimation and genomic prediction. Comparing to the GS performance of single-trait models, the prediction accuracy of nearly all traits could be improved by multi-trait models, which might because multiple-trait models used information from genetically correlated traits. Furthermore, we observed high significant negative correlation between the increased prediction accuracy from single-trait to multiple-trait models and estimated heritability. The results indicated that low-heritability traits could borrow more information from correlated traits and hence achieve higher prediction accuracy. The research first reported heritability estimation in rabbits by using genome-wide markers, and provided 50K as an optimal marker density for further microarray design, genetic evaluation and genomic selection in Angora rabbits. We expect that the work could provide strategies for GS in early selection, and optimize breeding programs in rabbits.

Characterization and functional analysis of SMAD2 regulation in hair follicle cycle in Angora rabbits.

Hair follicle (HF) development is characterized by periodic growth cycles regulated by numerous factors. We previously showed that SMAD2 might be involved in the HF growth cycle in Angora rabbits. However, its extra role in the HF growth and development remains obscure. In this study, we cloned the complete coding sequence (CDS) of the Angora rabbit SMAD2 gene. Within SMAD2 CDS, we identified the open reading frame (ORF) had a length of 1314 bp and encoding 437 amino acids. Bioinformatics analyses revealed that the SMAD2 protein is unstable and hydrophilic, and predominatelylocalizesin the cell nucleus. We identified that SMAD2 expression was elevated in the telogen phase of the during HF cycle. The knockdown and overexpression of SMAD2 could regulate HF growth and development related genes, such as WNT2, FGF2, and LEF1.Furthermore, SMAD2 may upregulate TGF-ß signaling pathway-related genes, including TFDP1, E2F4, and RBL1. In conclusion, our results indicate that SMAD2 plays a vital role in HF development by regulating the TGF-ß signaling pathway.

Characterization and functional analysis of Krtap11-1 during hair follicle development in Angora rabbits (Oryctolagus cuniculus).

BACKGROUND: Keratin-associated protein (KAP), the structural protein molecule of hair fibers, plays a key role in determining the physical properties of hair. Studies of Krtap11-1 have focused only on its localization. Functional studies of Krtap11-1 in hair follicle development have so far not been reported. OBJECTIVE: This study aimed to provide evidence for the role of Krtap11-1 in skin and hair development. METHODS: Full-length cloning and analysis of Krtap11-1 were conducted to ascertain its function. Overexpression vectors and interference sequences were constructed and transfected into RAB-9 cells. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to investigate the hair follicle developmental stage of Krtap11-1, the expression of different tissues, and the effects on other hair follicle development-related genes. RESULTS: The full length of cloned Krtap11-1 was 947 bp. Krtap11-1 was confirmed to be a hydrophilic protein localized mostly in mitochondria. The greatest mRNA expression was observed in skin. Using a follicle synchronization model, it was found that Krtap11-1 mRNA expression levels first increased then decreased over the passage of time, principally during hair follicle catagen and telogen. Following the overexpression of Krtap11-1, mRNA expression levels of the WNT-2, KRT17, BMP-2, and TGF-ß-1 genes increased, and LEF-1 decreased (P < 0.05), the converse after the corresponding use of si-RNA interference. CONCLUSIONS: Krtap11-1 exerts a promoting effect. The results provide novel insight into the relationship between hair follicle development and Krtap11-1 gene expression.