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Article

Identification of N6-Methyladenosine-Related Factors and the Prediction of the Regulatory Mechanism of Hair Follicle Development in Rex and Hycole Rabbits

by
Gang Luo
1,2,
Ruiguang Gong
1,
Yaotian Ai
1,
Tongyan Zhu
1 and
Zhanjun Ren
1,*
1
College of Animal Science and Technology, Northwest A&F University, Xianyang 712100, China
2
College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou 350000, China
*
Author to whom correspondence should be addressed.
Biology 2023, 12(11), 1448; https://doi.org/10.3390/biology12111448
Submission received: 31 August 2023 / Revised: 13 November 2023 / Accepted: 14 November 2023 / Published: 17 November 2023
(This article belongs to the Section Developmental and Reproductive Biology)
Browse Figures
Versions Notes

Abstract

:

Simple Summary

N6-methyladenosine (m6A) is an important modification for genes. Hair follicle development is crucial for the animal fur economy. To improve the quality of animal fur and solve the problem of baldness in people, we explored the regulatory mechanism of m6A on rabbit hair follicles and found that five methylases regulated the development of hair follicles through differential genes/signal pathways. These findings laid a molecular foundation for improving the quality of animal fur and solving the problem of baldness in people.

Abstract

Hair follicle development directly affects the development of the rabbit fur industry. The growth and development of a hair follicle is modified and regulated by many genes and mechanisms. M6A is an important RNA modification. However, there are few studies on the effects of the regulation of m6A on hair follicle growth and development. In this study, hematoxylin–eosin (HE) staining was used to explore the difference in hair follicle development between Rex rabbits and Hycole rabbits, and we performed m6A sequencing to identify the key genes with m6A modification in hair follicle growth. The results showed that the hair length, coarse hair percentage, primary hair follicle ratio, and skin thickness of Hycole rabbits were significantly higher than those of Rex rabbits. However, the proportion of secondary hair follicles in Hycole rabbits was significantly lower than that in Rex rabbits. In addition, we found five differential methylases, 20 differential genes, and 24 differential signaling pathways related to hair growth and development. The results of the Sankey diagram showed that 12 genes were related to 13 signal pathways. Finally, we found that five methylases regulated the development of hair follicles through differential genes/signal pathways. These findings laid a molecular foundation for the function of m6A modification in hair development.

1. Introduction

Rex rabbits are a type of rabbit with high hair density, capillary, and short hair. Hycole rabbits are a type of rabbit with low hair density and thick and long hair. The hair quality of rabbits is closely related to the hair density, which is mainly determined by the hair follicle density [1]. Therefore, the study of hair follicle development is of great significance to rabbit fur production. Hair follicle cycling is a complex biological process [2]. Hair follicles undergo anagen, catagen, and telogen cycles [3]. Many mRNAs and miRNAs are involved in the formation of hair follicles [4,5,6]. Hair follicle morphogenesis depends on many signaling pathways such as WNT, Shh, p53, TGF-β, Notch, and BMP [7]. However, the regulation pathways and methods of m6A modification on hair-follicle-development-related mRNAs in rabbits is unknown.
M6A is a common RNA modification [8]. At present, it has been found that m6A modification has been carried out under the action of methylase (METTL5, METTL14, METTL3, WTAP, and METTL4) and demethylase (FTO and ALKBH5) [9,10,11,12,13]. In addition, “reader” proteins containing YTHDC1, IGFBP2 and other YTH domain proteins are also determining factors in the m6A modification process [13,14,15,16,17,18]. These enzymes play an important role in animal growth [19], fat metabolism [20], reproduction [21], and other physiological processes.
In this study, we identified methylases, methylated genes, and signal pathways in Rex rabbit and Hycole rabbit skin by the MeRIP-seq method. Based on existing studies, we found five different methyltransferases, 20 methylation-modified differential genes, and 24 differential signaling pathways related to hair follicle development in Rex rabbits and Hycole rabbits. Finally, we found that 13 signal pathways were regulated by 12 genes among the genes and signal pathways we selected. In addition, we found that five methylases mediated 20 methylated genes to regulate hair follicle development through multiple pathways based on existing studies. This study lays a molecular theoretical foundation for further exploring the regulation of rabbit hair follicle development by m6A modification.

2. Material and Methods

2.1. Animals

Three newborn female Rex rabbits and three newborn female Hycole rabbits were used for methylated RNA immunoprecipitation sequencing (MeRIP-seq) [22]. Three 165-day-old Rex rabbits and three 165-day-old Hycole rabbits were used for photographing, hair index determination, and HE staining. All rabbits were collected from the same farm of the Northwest A&F University (Yangling, Shannxi, China). The rabbit farm belongs to Professor Ren Zhanjun, and he permitted the experiment.

2.2. Hair Index Determination

Pictures of Rex rabbits and Hycole rabbits were taken with a high pixel mobile phone (64 million pixels). The calculation method for the proportion of primary hair follicles was as follows: Firstly, we selected three 0.575 × 0.862 m2 microscope fields. Then, we recorded the number of hair follicles and obtained the number of primary hair follicles and all hair follicles per unit area. Finally, the proportion of primary hair follicles was calculated. We collected approximately 0.003 g of rabbit hair and recorded its weight as T1 (n = 3). The coarse wool was selected, and we recorded its weight as T2. Coarse wool ratio = T2/T1 × 100%. The hair length was directly measured by vernier caliper after collection.

2.3. Hematoxylin–Eosin (HE) Staining

A proper amount of rabbit back skin tissue was collected and fixed in formaldehyde solution. Then, the skin tissues were treated, embedded, sectioned and stained. The specific method is the same as that in the previous study [23]. Briefly, alcohol was used to remove the water in the tissue block, and then xylene was used to replace the alcohol in the tissue block. Tissue blocks were embedded in paraffin and cut into thin sections on a microtome. The slices were sequentially placed in xylene I (8 min), xylene II (8 min), anhydrous ethanol I (6 min), anhydrous ethanol II (6 min), 95% alcohol (6 min), 85% alcohol (6 min), and 75% alcohol (5 min) and rinsed with running water. Slices were stained with Harris hematoxylin for 3–8 min and rinsed with tap water. Then, the slices were differentiated with 1% hydrochloric acid alcohol for a few seconds and rinsed with tap water. The eosin staining solution was used to stain the slices for 1–3 min. Subsequently, we sequentially placed the slices in 75% alcohol (30 s), 85% alcohol (30 s), 95% alcohol I (1 min), 95% alcohol II (2 min), anhydrous ethanol I (5 min), anhydrous ethanol II (5 min), xylene I (5 min), and xylene II (7 min) to dehydrate and become transparent. Finally, we took the slices out of xylene and let them dry slightly; then, we sealed them with neutral gum. The primary hair follicle ratio and skin thickness were measured for each sample with a light microscope.

2.4. RNA Fragmentation

RNA was extracted from skin of rabbits by TRIzol reagent (Invitrogen Co., Carlsbad, CA, USA). Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) was used to assess the concentration and integrity of the RNA. The poly (A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, cat.e6150, Ipswich, MA, USA) under 86 °C, 7 min. Fragmentation buffer was used to break the mRNA into ∼100 nucleotides fragments.

2.5. M6A IP and Library Construction

The fragmented mRNA were divided into immunoprecipitation (IP) (95%) and IP control (input) groups (5%). We followed the instructions of the m6A RNA methylation library construction kit (A&D technology, Beijing, China) to IP RNA. Briefly, the cleaved RNA fragments were incubated for 2 h at 4 °C with m6A-specific antibody (No. 202003, Synaptic Systems, Göttingen, Germany) in IP buffer (50 mM Tris-HCl, 750 mM NaCl, and 0.5% Igepal CA-630). Then the IP RNA was reverse transcribed by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, Carlsbad, CA, USA). After, we performed the heat-labile UDG enzyme (NEB, cat.m0280, Ipswich, MA, USA) treatment of the U-labeled second-stranded DNAs. Then, the mRNA reacted with the antibody (binding to m6A modification site) with magnetic beads, and sequencing with high-throughput (Illumina Novaseq™6000) (LC-Bio Technology Co., Ltd., Hangzhou, China, 2020) was performed.

2.6. RT-qPCR

The Prime Script RT Reagent Kit (Takara Bio, Saint-Germain-en Laye, France) was used to reverse transcribe the total RNA. RT-qPCR experiments were carried out with a 10 µL system by SYBR Green. β-Actin was used as an internal control. All the primers used for qPCR are listed in Table 1.

2.7. Data Analysis

After sequencing, we used fastp (https://github.com/OpenGene/fastp, accessed on 29 May 2022) [24], the comparison tool bowtie2 [25], and HISAT2 (2.2.1.0) (http://daehwankimlab.github.io/hisat2, accessed on 24 July 2020) [26] software to filter, remove, and compare raw reads separately. The R-Pack exomepeak2 (https://bioconductor.org/packages/edgeR, accessed on 19 May 2022) [27] and DiffBind (3.5) [28] software were used to merge peaks between groups and calculate the abundance of peaks in each sample. StringTie (2.2.1) (https://ccb.jhu.edu/software/stringtie) was used to perform the expression level on 20 May 2022 for all mRNAs from Input libraries by calculating the FPKM (total exon fragments /mapped reads (millions) × exon length (kB)). The differentially expressed mRNAs were selected with log2 (fold change) > 1 or log2 (fold change) < −1 and p value < 0.05 by R package edgeR (4.0.1) (https://bioconductor.org/packages/edgeR on 20 May 2022). Genomes (KEGG) pathway analysis was performed using the database for annotation, visualization, and integrated discovery [29]. Hair and follicle data results were presented as the mean ± standard deviation (SD). GraphPad Prism7 (GraphPad Software, La Jolla, CA, USA) was used to assess the difference. The Student’s t-test was used to analyze the significance of the different levels.

3. Results

3.1. Difference in Hair Follicles between Rex and Hycole Rabbits

Based on the pictures of the Rex rabbits and Hycole rabbits, we found that the fur of the Rex rabbits was different from that of Hycole rabbits (Figure 1A). The results showed that the hair of the Hycole rabbits was significantly longer than that of Rex rabbits (p < 0.01) (Figure 1B). In addition. the coarse hair rate of the Hycole rabbits was also significantly higher than those of the Rex rabbits (p < 0.01) (Figure 1C). The proportion of primary hair follicles of the Rex rabbits was significantly lower than that of the Hycole rabbits (p < 0.01) (Figure 2A,B). At the same time, the primary hair follicle ratio of Hycole rabbits was significantly higher than that of the Rex rabbits, whereas the ratio of the secondary hair follicles of the Rex rabbits was significantly higher than that of the Hycole rabbits (p < 0.01) (Figure 2C). The results showed that the skin of the Rex rabbit was significantly thinner than that of the Hycole rabbit (p < 0.01) (Figure 2D,E).

3.2. Summary and Quality Control of Rabbit m6A Sequencing Data

As shown in Supplementary Table S1, MeRIP-seq produced 65,627,376–97,648,830 raw reads from input or IP skin tissues from Rex rabbit (ski) and Hycole rabbit (FYM). We found the GC content in the Rex rabbits’ IP and input was lower than that in the Hycole rabbits. In addition, the proportion of unique mapped reads was higher than 62.30%, and the proportion of multi-mapped reads varied from 3.27% to 25.64% (Supplementary Table S2).

3.3. General Features of Rabbit m6A Methylation

The samples were clustered by calculating the correlation coefficient between the Rex rabbit skin samples and the Hycole rabbit skin samples, which indicated good uniformity within the group (Figure 3A). As shown in Figure 3B, m6A-modified classical sequences RRACH appeared in the sequencing results of the Rex rabbits and Hycole rabbits. According to the statistics, 6093 peaks were methylated both in the Rex rabbit skin and the Hycole rabbit skin. Further, 3237 and 12,405 peaks were specifically methylated in the Rex rabbits’ skin and the Hycole rabbits’ skin, respectively (Figure 3C). To explore the preferential localization of m6A, we counted the distribution of peaks and found that in the CDS, the start and stop codons were the main areas of m6A (Figure 3D). In addition, the peaks enriched in the Rex rabbits were higher than those in the Hycole rabbits in the start codons (Figure 3D). However, the peaks enriched in the Rex rabbits were lower than those in Hycole rabbits in the stop codons (Figure 3D).

3.4. KEGG Pathway, Methylases, and Methylation Modifying Genes in Rex Rabbit Skin and Hycole Rabbit Skin

To predict the m6A-modified functions associated with hair follicle development, we analyzed the KEGG pathway and found there were 24 differential pathways involved in hair follicle development including Focal adhesion, Hippo, MAPK, WNT, cAMP, and other signaling pathways containing multiple genes (Figure 4). In addition, we found five differential methylation enzymes and 20 differential methylation modification genes were involved in hair follicle development based on the existing study results (Figure 5). To verify the results, we randomly selected two genes (IGF1 and EGFR) for RT-qPCR validation. The expression levels of the IGF1 gene and EGFR gene in the Rex rabbit skin were significantly higher than those in the Hycole rabbit skin (p < 0.01) (Figure 6). In addition, we found the distribution of the m6A modification of 20 genes on chromosomes was significantly different (Figure 7).

3.5. Regulation of Methylation Modified Genes by Methylase

As shown in Supplementary Figure S1, we found there were many m6A modification sites in 20 different genes. According to the existing research, YTHDC1 can directly regulate the expression of AKT (Figure 8). At the same time, METTL4 and IGF2BP2 can regulate AKT expression through PI3K and insulin, respectively. AKT can directly regulate the expression of CD34 and SOX9 and indirectly regulate the expression of the ETS1, TRPV3, HOXC13, FGF3, CD200, FGFR2, IGF1, LIPH, and LGR4 genes (Figure 8). Another regulatory pathway shows that IGF2BP2 can regulate the expression of the EGFR gene through insulin (Figure 8). As shown in Figure 8, METTL3 can indirectly regulate the expression of the RUNX2 and LEF1 genes. At the same time, METTL3 and METTL5 can regulate the expression of the WNT2, WNT10B, WNT5A, EDAR, BMP4, MSX2, LIPH, and LGR4 genes (Figure 8).

3.6. The Connection between Differential Pathways and Differential Genes

By drawing a Sankey diagram, we found SOX9, LGR4, and EDAR were important regulators in the cAMP, WNT, and NF-Kβ signaling pathways, respectively. BMP4, LEF1, FGF2, and FGFR2 were involved in the regulation of the two signaling pathways. In addition, WNT2, WNT10B, and WNT5A regulated three signal pathways. IGF1 and EGFR regulated six signal pathways (Figure 9). So, We selected IGF1 and EGFR, which had the most regulated signaling pathways, for PCR validation.

4. Discussion

A hair follicle is an important skin derivate with a unique structure and periodic regeneration ability, which plays an important role in hair growth [30]. A hair follicle mainly regulates the growth, color, and fixation of hair [2]. The development of hair follicles plays an important role in the fur rabbit industry [31]. We found that the fur of Rex rabbits was shorter and denser than that of Hycole rabbits. Studies have shown that single nucleotide deletion in exon 9 (1362delA) of LIPH is the reason for the hair phenotype of Rex rabbits [32]. The average hair length, the proportion of primary hair follicles, and the thickness of skin in Hycole rabbits were significantly higher than those in Rex rabbits. The diameter of the primary hair follicles and the volume of their papillae were significantly larger than those of he secondary hair follicles. However, the differentiation and proliferation of secondary hair follicles were faster, which is beneficial for temperature regulation. Therefore, Rex rabbits and Hycole rabbits are two ideal experimental animals to study the differences in hair volume, hair length, and hair follicle growth.
In order to study the mechanism of m6A modification on hair growth and development, we selected the skin tissues of Rex rabbits and Hycole rabbits for MeRIP-seq and found many differences in gene modification. Firstly, we found the typical m6A motif RRACH of animals and plants in two kinds of rabbits [33,34], and the motifs of the two kinds of rabbits are different at many gene loci in Figure 3B. We also found that the location and distribution of the peaks were significantly different in the two breeds of rabbits. Previous studies have shown that m6A methylation regulated the expression of genes related to cashmere growth [35]. These results suggested that m6A regulates hair development.
Based on the existing study results, we found that five methylases can regulate different genes through multiple pathways. METTL3 can directly regulate the expression of RUNX2 and LEF1 [36,37]. In addition, METTL3 and METTL5 both regulated the WNT signaling pathway [38,39]. WNT2, WNT5A, and WNT10B are key factors in the WNT signal pathway. Wnt/β-catenin acts on the upstream of BMP4 [40] and regulates EDAR and MSX2 through β-catenin and BMP4, respectively [41,42]. At the same time, MAPK regulates chondrocytes apoptosis through WNT/NF-κB pathways [43]. NF-κB plays a role in the expression of LGR4 and LIPH through microR-34c and miR-195-5p, respectively [44,45,46,47]. Knockdown of METTL4 led to downregulation and inactivation of the INSR pathway [48], thereby regulating the IRS-1/PI3K/AKT pathway to improve insulin resistance [49]. In addition, AKT regulates the expression of the ETSI [50], TRPV3 [51,52], HOXC13 [53,54,55], and FGF5 [56,57,58] genes through the mTOR, mTOR/TGF-β/miR-181, HIF-1a/miR-485-5p, and ERK1/2/LIN28/let-7b signaling pathways, respectively.
The functions of IGF2BP2 are associated with insulin resistance [59,60], and insulin regulates the EGFR gene to promote the migration of human corneal epithelial cells [61]. In addition, insulin can inhibit hepatic gluconeogenesis by activating the AKT/FOXO1 signaling pathway [62], and FOXO1 inhibits leptin regulation by blocking STAT3 interaction [63]. Leptin and estradiol interact to regulate the expression of IGF-1 [64]. In addition, FOXO1 transrepresses PPARγ transactivation to regulate miR-142-3p, and CD200 is a target gene of miR-142-3p [65,66,67]. At the same time, PPARγ regulates keratinocyte proliferation by targeting FGFR2 with miR-125b [68,69]. MiR-451a represses the AKT/mTOR signaling pathway [70], and AKT regulates SOX9 expression [71] to maintain the imatinib-resistant phenotype of CML CD34+ cells [72]. These existing studies have shown that five methylases can regulate the expression of 20 methylation-modified genes. However, the specific regulation mode is still unclear. This study provides a new possibility for further study on how the five methylases regulate 20 methylated-modified genes.
To further explore the regulation of methylation-modified genes on hair follicle growth and development, we associated methylation-modified genes with differential signaling pathways through a Sankey diagram. As shown in Figure 8, there were 12 genes that regulated 13 differential signaling pathways through different pathways. At the same time, we found that 13 signaling pathways played an important role in the growth and development of hair follicles and hairs. NF-kappaβ promotes hair follicle growth [73]. The FOXO signaling pathway mediates the changes in epidermal morphology, which is closely related to the development of hair follicles [74]. Focal Adhesion Signaling plays an important role in Cochlear Hair Cell Morphology [75]. Some canonical pathways such as WNT, MAPK, TGF-β, and Hippo signaling pathways were detected as promoting the hair follicle growth [76,77,78]. The PI3K/AKT [79], mTOR [79], JAK-STAT [80], Gap junction [81], and cAMP [82] signaling pathways are involved in the development of hair follicles and hairs. In addition, p53 regulated hair follicle regression [83]. So, 12 methylation modification genes regulate the development of hair follicles and hairs through 13 differential signaling pathways.

5. Conclusions

In summary, this study analyzed the modification of m6A methylation in Rex and Hycole rabbit skin tissue. We found five differential methylases, 20 differential genes, and 24 differential signaling pathways related to hair development in Rex and Hycole rabbits. Five methylases regulated the expression of 20 genes related to hair follicle development, of which 12 genes were found to regulate 13 important hair follicle signaling pathways. The development of hair follicles directly affected the growth and density of hair. Studying the effect of m6A on the development of hair follicles lays a theoretical foundation for m6A modification to regulate the development of rabbit hair follicles.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biology12111448/s1, Figure S1: The abundance of m6A in the Rex rabbit and Hycole rabbit skin; Table S1: Summary of reads quality control; Table S2: Summary of reads mapping to the rabbit reference genome.

Author Contributions

Conceptualization: Z.R. Formal analysis: G.L., T.Z. and Y.A. Resources: Z.R., Y.A. and R.G. Writing: G.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Innovation integration promotion of technological innovation for improving quality and efficiency in rabbit industry (NYKJ-2021-YL (XN)28v), the Research, development, demonstration and promotion of key technologies for large-scale and standardized healthy rabbit breeding (NYKJ-2020-YL-16v), the and Research and popularization of integrated nutrition technology for meat rabbit mother and calf (NYKJ-2021-YL (XN)26J).

Institutional Review Board Statement

This study was conducted in strict accordance with the ethical standards and approved by the Institutional Animal Care and Use Committee of the College of Animal Science and Technology, Northwest A&F University, Yangling on 15 October 2021, (Permit Number: No. DK-2019008).

Informed Consent Statement

Not applicable.

Data Availability Statement

Raw data have been uploaded to NCBI, which are deposited under SRA BioProject accession PRJNA863730.

Acknowledgments

We thank the staff at our laboratory for their ongoing assistance.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Hair difference between Rex Rabbit and Hycole rabbit. (A) Pictures of Rex rabbits and Hycole rabbits; (B) hair length of Rex rabbits and Hycole rabbits; (C) coarse wool ratio of Rex rabbits and Hycole rabbits (“**”, p ≤ 0.01).
Figure 1. Hair difference between Rex Rabbit and Hycole rabbit. (A) Pictures of Rex rabbits and Hycole rabbits; (B) hair length of Rex rabbits and Hycole rabbits; (C) coarse wool ratio of Rex rabbits and Hycole rabbits (“**”, p ≤ 0.01).
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Figure 2. Hair follicle distribution and skin thickness. (A) HE staining cross section of skin; (B) proportion of primary hair follicles; (C) primary and secondary follicle density; (D) HE staining longitudinal section of skin; (E) skin thickness (longitudinal section of skin in Rex rabbits and Hycole rabbits) (“**”, p ≤ 0.01).
Figure 2. Hair follicle distribution and skin thickness. (A) HE staining cross section of skin; (B) proportion of primary hair follicles; (C) primary and secondary follicle density; (D) HE staining longitudinal section of skin; (E) skin thickness (longitudinal section of skin in Rex rabbits and Hycole rabbits) (“**”, p ≤ 0.01).
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Figure 3. (A) Cluster analysis heat map; (B) sequence logo identified from Rex rabbit and Hycole rabbit skin; (C) overlap of m6A peaks from Rex rabbit and Hycole rabbit skin; (D) distribution of m6A peaks across the length of mRNA.
Figure 3. (A) Cluster analysis heat map; (B) sequence logo identified from Rex rabbit and Hycole rabbit skin; (C) overlap of m6A peaks from Rex rabbit and Hycole rabbit skin; (D) distribution of m6A peaks across the length of mRNA.
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Figure 4. Enrichment pathway of the m6A peak related to hair growth and development.
Figure 4. Enrichment pathway of the m6A peak related to hair growth and development.
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Figure 5. (A) Cluster heat map of differential methylases from Rex rabbit and Hycole rabbit skin; (B) cluster heat map of differential genes related to hair development from Rex rabbit and Hycole rabbit skin.
Figure 5. (A) Cluster heat map of differential methylases from Rex rabbit and Hycole rabbit skin; (B) cluster heat map of differential genes related to hair development from Rex rabbit and Hycole rabbit skin.
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Figure 6. (A) IGF1 expression level in Rex rabbit and Hycole rabbit skin; (B) EGFR expression level in Rex rabbit and Hycole rabbit skin; (“**”, p ≤ 0.01).
Figure 6. (A) IGF1 expression level in Rex rabbit and Hycole rabbit skin; (B) EGFR expression level in Rex rabbit and Hycole rabbit skin; (“**”, p ≤ 0.01).
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Figure 7. Distribution of differential genes on the chromosomes of 20 genes’ m6A modification.
Figure 7. Distribution of differential genes on the chromosomes of 20 genes’ m6A modification.
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Figure 8. Pathway map of key genes regulated by five methylases.
Figure 8. Pathway map of key genes regulated by five methylases.
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Figure 9. Sankey diagram of genes and signal pathways.
Figure 9. Sankey diagram of genes and signal pathways.
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Table 1. Primers used in this study.
Table 1. Primers used in this study.
Gene NamePrimer Sequence (5′-3′)Tm (°C)
β-actinGGAGATCGTGCGGGACAT60
GTTGAAGGTGGTCTCGTGGAT
IGF1ACCCACCCTAACCTGCTGTA60
TCCTGTGGGCTTGTTGAAAT
EGFRACCTTGTCATTCAGGGGGATG60
ACACAAGCCATGGTGGAACT
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Luo, G.; Gong, R.; Ai, Y.; Zhu, T.; Ren, Z. Identification of N6-Methyladenosine-Related Factors and the Prediction of the Regulatory Mechanism of Hair Follicle Development in Rex and Hycole Rabbits. Biology 2023, 12, 1448. https://doi.org/10.3390/biology12111448

AMA Style

Luo G, Gong R, Ai Y, Zhu T, Ren Z. Identification of N6-Methyladenosine-Related Factors and the Prediction of the Regulatory Mechanism of Hair Follicle Development in Rex and Hycole Rabbits. Biology. 2023; 12(11):1448. https://doi.org/10.3390/biology12111448

Chicago/Turabian Style

Luo, Gang, Ruiguang Gong, Yaotian Ai, Tongyan Zhu, and Zhanjun Ren. 2023. "Identification of N6-Methyladenosine-Related Factors and the Prediction of the Regulatory Mechanism of Hair Follicle Development in Rex and Hycole Rabbits" Biology 12, no. 11: 1448. https://doi.org/10.3390/biology12111448

APA Style

Luo, G., Gong, R., Ai, Y., Zhu, T., & Ren, Z. (2023). Identification of N6-Methyladenosine-Related Factors and the Prediction of the Regulatory Mechanism of Hair Follicle Development in Rex and Hycole Rabbits. Biology, 12(11), 1448. https://doi.org/10.3390/biology12111448

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