Chicken Muscle Development Research Articles

METTL16 inhibits differentiation and promotes proliferation and slow myofibers formation in chicken myoblasts

N6-methyladenosine (m6A) plays a crucial regulatory role in muscle growth and development. In our previous studies, we identified a m6A methyltransferase, Methyltransferase like 16 (METTL16), which is associated with chicken muscle development and muscle fiber type conversion. To further understand the regulatory role of METTL16 in chicken muscle function, we analyzed its expression in muscle tissues with different myofiber type compositions and in chicken primary myoblasts (CPMs) at various stages. We also manipulated METTL16 expression in CPMs to examine its effects on cell proliferation, differentiation, muscle fiber type formation, and global m6A RNA methylation status. Our results showed that METTL16 expression increased during myoblast proliferation and gradually decreased in the late differentiation stage. Furthermore, METTL16 exhibited specific expression in slow-twitch muscles. Cell Counting Kit-8 assays, 5-Ethynyl-2'-deoxyuridine staining, RT-qPCR, Western blot, and immunofluorescence analyses showed that METTL16 promotes myoblast proliferation while inhibiting myoblast differentiation. We also observed that METTL16 induces the upregulation of slow-twitch myosin heavy chain (MyHC) and slow-twitch-specific genes in myotubes, while downregulating fast-twitch MyHC and fast-twitch-specific genes. Furthermore, both interference and overexpression of METTL16 led to changes in overall cellular m6A modification levels and Methyltransferase like 3 (METTL3) expression levels. These findings confirm that METTL16 plays a key role in myoblast proliferation, differentiation, and muscle fiber type formation in chickens. Considering the role of myoblasts in chicken muscle growth and meat quality regulation, METTL16 may serve as a key target for molecular selection in chicken meat traits.

In ovo feeding of l-arginine and selenium nanoparticles influences post-hatch growth, muscle development, antioxidant status, and meat quality in slow-growing chickens.

This study investigated the effects of in ovo feeding (IOF) of l-arginine (L-Arg), selenium nanoparticles (SeNP), and a combination of L-Arg and SeNP on the hatchability, post-hatch growth, muscle development, antioxidant status, and meat quality of slow-growing chickens. On day 18 of incubation, a total of 960 fertilized eggs with similar weights were randomly assigned to 4 treatment groups with 4 replicates of 60 eggs each: (1) non-injected control group (Control), (2) injected with 1% of L-Arg (IOF_L-Arg), (3) injected with 0.3 µg/egg of SeNP (IOF_SeNP), and (4), injected with 1% of L-Arg and 0.3 µg/egg of SeNP (IOF_L-Arg + SeNP). A completely randomized design was used. After hatching, 640 mixed-sex chicks were allocated to 4 treatment groups and split into 4 replicate pens (40 birds per pen). All groups of chicks were fed with commercial feed ad libitum until they reached 63 d of age and were subsequently weighed and slaughtered. The results of the present study showed that hatchability was similar among treatments. Final BW or breast muscle yield was not affected (P > 0.05) by IOF treatment. Chickens treated with IOF_L-Arg + SeNP exhibited decreased feed conversion ratio, drip loss, and increased protein content in breast meat (P < 0.05). The IOF_L-Arg + SeNP group exhibited a higher density of breast muscle fibers than the control group (P < 0.05). Overall, in ovo feeding of L-Arg combined with SeNP resulted in improved feed efficiency and enhanced antioxidant capacity at hatch without any adverse effects on chicken hatchability, health, or subsequent growth. Furthermore, meat from chickens in the IOF_L-Arg + SeNP group exhibited a preferable texture with a higher protein content.

Dynamic Transcriptome Profile Analysis of Mechanisms Related to Melanin Deposition in Chicken Muscle Development.

The pectoral muscle is an important component of skeletal muscle. The blackness of pectoral muscles can directly affect the economic value of black-boned chickens. Although the genes associated with melanogenesis in mammals and birds have been thoroughly investigated, only little is known about the key genes involved in muscle hyperpigmentation during embryonic development. Here, we analyzed melanin deposition patterns in the pectoral muscle of Yugan black-boned chickens and compared differentially expressed genes (DEGs) between the muscles of Wenchang (non-black-boned chickens) and Yugan black-boned chickens on embryonic days 9, 13, 17, and 21. Melanin pigments were found to gradually accumulate in the muscle fibers over time. Using RNA-seq, there were 40, 97, 169, and 94 genes were identified as DEGs, respectively, between Yugan black-boned chicken muscles and Wenchang chickens at embryonic day 9, 13, 17, and 21 stages (fold change ≥2.0, false discovery rate (FDR) < 0.05). Thirteen DEGs, such as MSTRG.720, EDNRB2, TYRP1, and DCT, were commonly identified among the time points observed. These DEGs were mainly involved in pigmentation, melanin biosynthetic and metabolic processes, and secondary metabolite biosynthetic processes. Pathway analysis of the DEGs revealed that they were mainly associated with melanogenesis and tyrosine metabolism. Moreover, weighted gene co-expression network analysis (WGCNA) was used to detect core modules and central genes related to melanogenesis in the muscles of black-boned chickens. A total of 24 modules were identified. Correlation analysis indicated that one of them (the orange module) was positively correlated with muscle pigmentation traits (r > 0.8 and p < 0.001). Correlations between gene expression and L* values of the breast muscle were investigated in Yugan and Taihe black-boned chickens after hatching. The results confirmed that EDNRB2, GPNMB, TRPM1, TYR, and DCT expression levels were significantly associated with L* values (p < 0.01) in black-boned chickens (p < 0.05). Our results suggest that EDNRB2, GPNMB, TRPM1, TYR, and DCT are the essential genes regulating melanin deposition in the breast muscle of black-boned chickens. MSTRG.720 is a potential candidate gene involved in melanin deposition in the breast muscles of Yugan black-boned chickens.

Identification of key differentially methylated genes regulating muscle development in chickens: insights from Jingyuan breed

Skeletal muscle development is a complex, regulated physiological process that involves myoblast proliferation and differentiation and the fusion of myotubes. In this study, phenotypic differences in the breast and leg muscles of 180-day-old Jingyuan chickens were investigated. Differentially methylated genes (DMG) that regulate muscle development were identified through differential expression analysis and weighted gene co-expression network analysis. Moreover, myoblasts were used as test material and treated with cycloleucine to investigate the effect of N6-methyladenosine (m6A) modification on their proliferation and differentiation. The results revealed that the myofiber diameter and cross-sectional area in the breast muscle of Jingyuan chickens were significantly smaller than those in the leg muscle, while myofiber density in the breast muscle was significantly higher. A total of 484 DMG were identified in both muscle types. Module gene association analysis with DMGs revealed multiple DMG associated with muscle development. In vitro cell model analysis revealed that cycloleucine treatment significantly downregulated the m6A modification level of myoblasts and inhibited their proliferation and differentiation. Additionally, stage-specific differences in LDHA, LDHB, and GAPDH expressions were observed during myoblast differentiation. Cycloleucine treatment significantly inhibited LDHA, LDHB, and GAPDH expression. These findings indicate that m6A methylation modifications play significant regulatory roles in muscle development, with LDHA, LDHB, and GAPDH being potential candidate genes for regulating muscle development. This study provides an essential theoretical basis for further study on the functional mechanisms of m6A modifications involved in muscle development.

Influence of incubation temperature on embryonic Days 4–7 on gene expression, M. pectoralis and M. gastrocnemius microstructure, and tibia characteristics in Ross 308 Broilers

ABSTRACT The incubation temperature can influence chicken bone and muscle development, and breast and leg muscles may respond differently to changes therein. Ross 308 broiler eggs were incubated at 36.5°C, 37.5°C (control), 38.5°C and 39.0°C on embryonic days 4–7. M. pectoralis and M. gastrocnemius were sampled at hatching and 5 weeks post-hatching, and differences in gene expression, microstructure and energy metabolism were examined. Tibia strength was tested, and gait scoring was performed. Broiler performance was similar at slaughter age, but initial growth was slowed by the 36.5°C treatment. The 38.5°C treatment reduced the M. gastrocnemius weight and shifted the M. pectoralis/M. gastrocnemius ratio. All treatments reduced M. gastrocnemius glycogen content relative to the control. The expression of single myogenesis-related genes was altered in the M. pectoralis by 36.5°C. The results indicate that the incubation temperature influences broiler muscle energy metabolism, and that the temperature of 38.5°C hampers leg muscle development.

Asparagine synthetase regulates the proliferation and differentiation of chicken skeletal muscle satellite cells.

Asparagine synthetase (ASNS) is an aminotransferase responsible for the biosynthesis of aspartate by using aspartic acid and glutamine. ASNS is highly expressed in fast-growing broilers, but few studies have reported the regulatory role of ASNS in muscle development. To explore the function of ASNS in chicken muscle development, the expression of ASNS in different chicken breeds and tissues were first performed by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). Then, using real-time quantitative RT-PCR, western blot, EdU assay, cell cycle assay and immunofluorescence, the effects of ASNS on the proliferation and differentiation of chicken skeletal muscle satellite cell (SMSC) were investigated. Finally, potential mechanisms by which ASNS influences chicken muscle fiber differentiation were identified through RNA-Seq. The mRNA expression pattern of ASNS in muscles mirrors trends in muscle fiber cross-sectional area, average daily weight gain, and muscle weight across different breeds. ASNS knockdown inhibited SMSC proliferation, while overexpression showed the opposite. Moreover, ASNS attenuated SMSC differentiation by activating the Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway. Additionally, 5-aminoimidazole-4-carboxamide1-β-D-ribofuranoside (AICAR) treatment suppressed the cell differentiation induced by siRNA-ASNS. RNA-Seq identified 1968 differentially expressed genes (DEGs) during chicken SMSC differentiation when overexpression ASNS. Gene Ontology (GO) enrichment analysis revealed that these DEGs primarily participated in 8 biological processes, 8 cellular components, and 4 molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified several significantly enriched signaling pathways, such as the JAK-STAT signaling pathway, TNF signaling pathway, Toll-like receptor signaling pathway, and PI3K-Akt signaling pathway. ASNS promotes proliferation while inhibits the differentiation of chicken skeletal muscle satellite cells. This study provides a theoretical basis for studying the role of ASNS in muscle development.

Transcriptome sequencing reveals the potential mechanisms of dietary lutein regulation on chicken leg muscle development

Lutein is an antioxidant that can indicate the oxidative status of organisms through its coloration and may be involved in the development process of chicken skeletal muscle. In this study, after feeding Nanhai Yellow Chickens with lutein-containing feed for 21 d, the lutein group significantly increased the muscle fiber diameter and decreased the fiber density in the chicken's leg muscles compared to the control group. To elucidate the potential regulatory mechanisms by which lutein is involved in muscle development, RNA-seq was used to detect changes in gene expression in chicken leg muscle tissue. After data analysis, a total of 249 significantly differentially expressed genes (DEG) were identified, including TGF-β superfamily (MSTN and TGFB1) and nonreceptor tyrosine kinase c-Src (SRC). Results from GO and KEGG analysis showed that the DEGs were enriched in GO terms such as positive regulation of the ERK1/ERK2 cascade and negative regulation of myoblast differentiation, as well as signaling pathways including the Toll-like receptor signaling pathway and the MAPK signaling pathway. These significantly enriched GO terms and pathways are closely related to muscle development, suggesting that lutein may play an important role in the process of chicken muscle development. This study provides insights into the regulatory mechanisms of dietary lutein on chicken muscle development.

Comparative analysis of the growth differences between hybrid Ningdu Yellow chickens and their parentals

Although the local high quality chicken breed in China has excellent flavor, its growth rate is inferior to that of foreign breeds. To improve the growth rate of local chicken breeds, it is crucial to study the mechanism of chicken muscle development. Herein, Ningdu Yellow chicken was used as the research object, and a new hybrid breed (W) was obtained by crossing the G, H and D lines, which combined the excellent physiological characteristics of its parents. Combined analysis of Ningdu Yellow chickens and their parents was carried out. Chickens from 105-day-old lines (W, G, H) were selected, and breast meat and serum were extracted for transcriptome sequencing and metabolome determination to study their growth differences. The live weight, carcass weight, half-eviscerated weight, eviscerated weight, and breast muscle weight of W were significantly higher than those of G and H. Differential expression analysis identified 1700 differentially expressed genes (DEG), and gene ontology and kyoto encyclopedia of genes and genomes (KEGG) analyses identified 33 and 1 pathways related to growth and development and steroid biosynthesis, respectively. Next, pairwise analysis identified 57 KEGG pathways, among which the MAPK signaling, steroid hormone biosynthesis, tight junction, and PPAR signaling pathways were involved in growth and development. Cluster analysis found that genes highly expressed in the W group were associated with regulation of the actin cytoskeleton, riboflavin metabolism, steroid biosynthesis, and glycerophospholipid metabolism. The top 2 clusters obtained by protein-protein interaction analysis were important for the growth and development of chickens. Finally, the metabolomic analysis found key differentially accumulated metabolites (DAM) that might be account for the growth differences. Further integrated analysis identified key DEGs and DAMs that might be responsible for the observed growth differences. This study identified genes governing growth traits in Ningdu Yellow chickens, laying a theoretical foundation for the development of chicken breeding, the utilization of hybrid supporting lines, and promotion of the Chinese chicken industry.

Genome-wide mapping of the binding sites of myocyte enhancer factor 2A in chicken primary myoblasts

Myocyte enhancer factor 2A (MEF2A) is a transcription factor that plays a critical role in cell proliferation, differentiation and apoptosis. In contrast to the wide characterization of its regulation mechanism in mammalian skeletal muscle, its role in chickens is limited. Especially, its wide target genes remain to be identified. Therefore, we utilized Cleavage Under Targets and Tagmentation (CUT&Tag) technology to reveal the genome-wide binding profile of MEF2A in chicken primary myoblasts thus gaining insights into its potential role in muscle development. Our results revealed that MEF2A binding sites were primarily distributed in intergenic and intronic regions. Within the promoter region, although only 8.87% of MEF2A binding sites were found, these binding sites were concentrated around the transcription start site (TSS). Following peak annotation, a total of 1903 genes were identified as potential targets of MEF2A. Gene Ontology (GO) enrichment analysis further revealed that MEF2A target genes may be involved in the regulation of embryonic development in multiple organ systems, including muscle development, gland development, and visual system development. Moreover, a comparison of the MEF2A target genes identified in chicken primary myoblasts with those in mouse C2C12 cells revealed 388 target genes are conserved across species, 1515 target genes are chicken specific. Among these conserved genes, ankyrin repeat and SOCS box containing 5 (ASB5), transmembrane protein 182 (TMEM182), myomesin 2 (MYOM2), leucyl and cystinyl aminopeptidase (LNPEP), actinin alpha 2 (ACTN2), sorbin and SH3 domain containing 1 (SORBS1), ankyrin 3 (ANK3), sarcoglycan delta (SGCD), and ORAI calcium release-activated calcium modulator 1 (ORAI1) exhibited consistent expression patterns with MEF2A during embryonic muscle development. Finally, TMEM182, as an important negative regulator of muscle development, has been validated to be regulated by MEF2A by dual-luciferase and quantitative real-time PCR (qPCR) assays. In summary, our study for the first time provides a wide landscape of MEF2A target genes in chicken primary myoblasts, which supports the active role of MEF2A in chicken muscle development.

High Muscle Expression of IGF2BP1 Gene Promotes Proliferation and Differentiation of Chicken Primary Myoblasts: Results of Transcriptome Analysis.

Muscle development is a multifaceted process influenced by numerous genes and regulatory networks. Currently, the regulatory network of chicken muscle development remains incompletely elucidated, and its molecular genetic mechanisms require further investigation. The Longsheng-Feng chicken, one of the elite local breeds in Guangxi, serves as an excellent resource for the selection and breeding of high-quality broiler chickens. In this study, we conducted transcriptome sequencing of the pectoral muscles of Longsheng-Feng chickens and AA broiler chickens with different growth rates. Through comprehensive bioinformatics analysis, we identified differentially expressed genes that affect muscle growth and showed that IGF2BP1 is a key participant in chicken muscle development. Subsequently, we employed QRT-PCR, EdU staining, and flow cytometry to further investigate the role of IGF2BP1 in the proliferation and differentiation of chicken myogenic cells. We identified 1143 differentially expressed genes, among which IGF2BP1 is intimately related to the muscle development process and is highly expressed in muscle tissues. Overexpression of IGF2BP1 significantly promotes the proliferation and differentiation of chicken primary myoblasts, while knockdown of IGF2BP1 significantly inhibits these processes. In summary, these results provide valuable preliminary insights into the regulatory roles of IGF2BP1 in chicken growth and development.

Novel Circular RNA CircSLC2A13 Regulates Chicken Muscle Development by Sponging MiR-34a-3p.

The skeletal muscle is the major muscle tissue in animals, and its production is subject to a complex and strict regulation. The proliferation and differentiation of myoblasts are important factors determining chicken muscle development. Circular RNAs (circRNAs) are endogenous RNAs that are widely present in various tissues of organisms. Recent studies have shown that circRNA plays key roles in the development of skeletal muscles. The solute carrier (SLC) family functions in the transport of metabolites such as amino acids, glucose, nucleotides, and essential nutrients and is widely involved in various basic physiological metabolic processes within the body. In this study, we have cloned a novel chicken circular RNA circSLC2A13 generated from the solute carrier family 2 member 13 gene (SLC2A13). Also, circSLC2A1 was confirmed by sequencing verification, RNase R treatment, and reverse transcription analysis. Currently, our results show that circSLC2A13 promoted the proliferation and differentiation of chicken myoblasts. The double luciferase reporter system revealed that circSLC2A13 regulated the proliferation and differentiation of myoblasts by competitive binding with miR-34a-3p. In addition, results indicated that circSLC2A13 acts as a miR-34a-3p sponge to relieve its inhibitory effect on the target SMAD3 gene. In summary, this study found that chicken circSLC2A13 can bind to miR-34a-3p and weaken its inhibitory effect on the SMAD family member 3 gene (SMAD3), thereby promoting the proliferation and differentiation of myoblasts. This study laid foundations for broiler industry and muscle development research.

Transcriptome analysis of long non-coding RNA associated with embryonic muscle development in chickens

ABSTRACT 1. Skeletal muscle is an important component of chicken carcass. In chickens, the number of muscle fibres is fixed during the embryonic period, and muscle development during the embryonic period determines the muscle development potential after hatching. 2. Beijing-You (BY) and Cornish (CN) chickens show completely different growth rates and body types, and two breeds were used in this study to explore the role of lncRNAs in muscle development during different chicken embryonic periods. A systematic analysis of lncRNAs and mRNAs were conducted in the pectoral muscle tissues of BY and CN chickens at embryonic days 11 (ED11), 13 (ED13), 15 (ED15), 17 (ED17), and 1-day-old (D1) using RNA-seq. A total of 4,104 differentially expressed transcripts (DETs) were identified among the five stages, including 2,359 lncRNAs and 1,745 mRNAs. 3. The number of DETs between the two breeds at ED17 (1,658 lncRNAs and 1,016 mRNAs) was much higher than the total number of DET at all the other stages (692 lncRNAs and 729 mRNAs), indicating that the two breeds show the largest difference in gene regulation at ED17. 4. Correlation analysis was performed for all differentially expressed lncRNAs and mRNAs during the five periods. Forty-three, cis interaction pairs of lncRNA-mRNA related to chicken muscle development were predicted. The expression of four pairs was verified, and the results showed MSTRG.12395.2-FGFBP2 and MSTRG.18590.6-FMOD were significantly up-regulated in CN at ED11 compared to BY and might be important candidate genes for embryonic muscle development.

Analysis of microRNA Expression Profiles in Broiler Muscle Tissues by Feeding Different Levels of Guanidinoacetic Acid.

The aim of this study was to explore the molecular mechanisms through which different levels of GAA affect chicken muscle development by influencing miRNA expression, to lay a theoretical foundation for the identification of key functional small RNAs related to poultry muscle development, and to provide new insights into the regulatory mechanisms of GAA on muscle development and meat quality in broilers. It provides a new theoretical basis for using GAA as a feed additive to improve feed performance. Small RNA sequencing technology was utilized to obtain the expression profiles of miRNA in the broiler pectoral muscle fed with different levels of GAA (0 g/kg, 1.2 g/kg and 3.6 g/kg). An analysis of differentially expressed miRNAs revealed 90 such miRNAs in the three combination comparisons, with gga-miR-130b-5p exhibiting significant differences across all three combinations. Furthermore, three of the differentially expressed miRNAs were performed by RT-qPCR verification, yielding results consistent with those obtained from small RNA sequencing. Target gene prediction, as well as the GO and KEGG enrichment analysis of differentially expressed miRNAs, indicated their involvement in muscle cell differentiation and other processes, particularly those associated with the MAPK signaling pathway. This study has, thus, provided valuable insights and resources for the further exploration of the miRNA molecular mechanism underlying the influence of guanidine acetic acid on broiler muscle development. Combined with previous studies and small RNA sequencing, adding 1.2 g/kg GAA to the diet can better promote the muscle development of broilers.

Identification of key differentially methylated genes in regulating muscle development and intramuscular fat deposition in chickens

Muscle development and intramuscular fat (IMF) deposition are intricate physiological processes characterized by multiple gene expressions and interactions. In this research, the phenotypic variations in the breast muscle of Jingyuan chickens were examined at three different time points: 42, 126, and 180 days old. Differential expression analysis and weighted gene co-expression network analysis (WGCNA) were performed to identify differentially methylated genes (DMGs) responsible for regulating muscle development and IMF deposition. The findings indicate a significant increase in breast muscle weight (BMW), myofiber diameter, and cross-sectional area, as well as IMF content, in correlation with the progressive number of growing days in Jingyuan chickens. The findings also revealed that 380 hypo-methylated and 253 hyper-methylated DMGs were identified between the three groups of breast muscle. Module gene and DMG association analysis identified m6A methylation-mediated multiple DMGs associated with muscle development and fat metabolism. In vitro cell modeling analysis reveals stage-specific differences in the expression of CUBN, MEGF10, BOP1, and BMPR2 during the differentiation of myoblasts and intramuscular preadipocytes. Cycloleucine treatment significantly inhibited the expression levels of CUBN, BOP1, and BMPR2, and promoted the expression of MEGF10. These results suggest that m6A methylation-mediated CUBN, MEGF10, BOP1, and BMPR2 can serve as potential candidate genes for regulating muscle development and IMF deposition, and provide an important theoretical basis for further investigation of the functional mechanism of m6A modification involved in adipogenesis.

3D genomic alterations during development of skeletal muscle in chicken1

The development of skeletal muscle are complicated processes involving genes responsible for proper muscle morphology, contractility, cell proliferation, differentiation, interactions, migration, and death. The three-dimensional chromatin architecture of skeletal muscle development has not been studied intensively although dynamic transcriptional regulation during differentiation of muscle cells is one of the most deeply studied processes. The RNA-seq was used to analyze the transcriptome pattern during chicken muscle development across 12 stages. Hi-C was used to build a chromatin architectures during four representative stages. ChIP-seq was conducted to identify enhancers in these four stages, which are occupied by histone H3K27ac and H3K4me3 peaks. Results show that large-scale genome architecture changes are mostly unidirectional, and coupled by complex on/off dynamic patterns of gene expression. Specifically, we observed 258.30 Mb of the genome undergoing A/B compartment switching. Notable alterations (316.57 Mb) of interaction frequencies within TADs were observed. Substantial aging-associated genes exhibited ascending connectivity with the compartment transition from repressive to active status during muscle development. Some muscle-related gene promoters that interacted with active enhancers during development, and some myopathy/aging-associated genes that were activated in aging muscle were founded. These results provide key insights into skeletal muscle development in vivo, and offer a valuable resource that allows in-depth functional characterization of candidate genes.

MYH1G-AS is a chromatin-associated lncRNA that regulates skeletal muscle development in chicken

BackgroundSkeletal muscle development is pivotal for animal growth and health. Recently, long noncoding RNAs (lncRNAs) were found to interact with chromatin through diverse roles. However, little is known about how lncRNAs act as chromatin-associated RNAs to regulate skeletal muscle development. Here, we aim to investigate the regulation of chromatin-associated RNA (MYH1G-AS) during skeletal muscle development.MethodsWe provided comprehensive insight into the RNA profile and chromatin accessibility of different myofibers, combining RNA sequencing (RNA-seq) with an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). The dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were used to analyze the transcriptional regulation mechanism of MYH1G-AS. ALKBH5-mediated MYH1G-AS N6-methyladenosine (m6A) demethylation was assessed by a single-base elongation and ligation-based qPCR amplification method (SELECT) assay. Functions of MYH1G-AS were investigated through a primary myoblast and lentivirus/cholesterol-modified antisense oligonucleotide (ASO)-mediated animal model. To validate the interaction of MYH1G-AS with fibroblast growth factor 18 (FGF18) protein, RNA pull down and an RNA immunoprecipitation (RIP) assay were performed. Specifically, the interaction between FGF18 and SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (SMARCA5) protein was analyzed by coimmunoprecipitation (Co-IP) and a yeast two-hybrid assay.ResultsA total of 45 differentially expressed (DE) lncRNAs, with DE ATAC-seq peaks in their promoter region, were classified as open chromatin-associated lncRNAs. A skeletal muscle-specific lncRNA (MSTRG.15576.9; MYH1G-AS), which is one of the open chromatin-associated lncRNA, was identified. MYH1G-AS transcription is coordinately regulated by transcription factors (TF) SMAD3 and SP2. Moreover, SP2 represses ALKBH5 transcription to weaken ALKBH5-mediated m6A demethylation of MYH1G-AS, thus destroying MYH1G-AS RNA stability. MYH1G-AS accelerates myoblast proliferation but restrains myoblast differentiation. Moreover, MYH1G-AS drives a switch from slow-twitch to fast-twitch fibers and causes muscle atrophy. Mechanistically, MYH1G-AS inhibits FGF18 protein stabilization to reduce the interaction of FGF18 to SMARCA5, thus repressing chromatin accessibility of the SMAD4 promoter to activate the SMAD4-dependent pathway.ConclusionsOur results reveal a new pattern of the regulation of lncRNA expression at diverse levels and help expound the regulation of m6A methylation on chromatin status.Graphical

Genome-wide characteristics and potential functions of circular RNAs from the embryo muscle development in Chengkou mountain chicken.

The Chengkou mountain chicken, a native Chinese poultry breed, holds significant importance in the country's poultry sector due to its delectable meat and robust stress tolerance. Muscle growth and development are pivotal characteristics in poultry breeding, with muscle fiber development during the embryonic period crucial for determining inherent muscle growth potential. Extensive evidence indicates that non-coding RNAs (ncRNAs) play a regulatory role in muscle growth and development. Among ncRNAs, circular RNAs (circRNAs), characterized by a closed-loop structure, have been shown to modulate biological processes through the regulation of microRNAs (miRNAs). This study seeks to identify and characterize the spatiotemporal-specific expression of circRNAs during embryonic muscle development in Chengkou mountain chicken, and to construct the potential regulatory network of circRNAs-miRNA-mRNAs. The muscle fibers of HE-stained sections became more distinct, and their boundaries were more defined over time. Subsequent RNA sequencing of 12 samples from four periods generated 9,904 novel circRNAs, including 917 differentially expressed circRNAs. The weighted gene co-expression network analysis (WGCNA)-identified circRNA source genes significantly enriched pathways related to cell fraction, cell growth, and muscle fiber growth regulation. Furthermore, a competitive endogenous RNA (ceRNA) network constructed using combined data of present and previous differentially expressed circRNAs, miRNA, and mRNA revealed that several circRNA transcripts regulate MYH1D, MYH1B, CAPZA1, and PERM1 proteins. These findings provide insight into the potential pathways and mechanisms through which circRNAs regulate embryonic muscle development in poultry, a theoretical support for trait improvement in domestic chickens.

RRM2 promotes the proliferation of chicken myoblasts, inhibits their differentiation and muscle regeneration

During myogenesis and regeneration, the proliferation and differentiation of myoblasts play key regulatory roles and may be regulated by many genes. In this study, we analyzed the transcriptomic data of chicken primary myoblasts at different periods of proliferation and differentiation with protein‒protein interaction network, and the results indicated that there was an interaction between cyclin-dependent kinase 1 (CDK1) and ribonucleotide reductase regulatory subunit M2 (RRM2). Previous studies in mammals have a role for RRM2 in skeletal muscle development as well as cell growth, but the role of RRM2 in chicken is unclear. In this study, we investigated the effects of RRM2 on skeletal muscle development and regeneration in chickens in vitro and in vivo. The interaction between RRM2 and CDK1 was initially identified by co-immunoprecipitation and mass spectrometry. Through a dual luciferase reporter assay and quantitative real-time PCR, we identified the core promoter region of RRM2, which is regulated by the SP1 transcription factor. In this study, through cell counting kit-8 assays, 5-ethynyl-2'-deoxyuridine incorporation assays, flow cytometry, immunofluorescence staining, and Western blot analysis, we demonstrated that RRM2 promoted the proliferation and inhibited the differentiation of myoblasts. In vivo studies showed that RRM2 reduced the diameter of muscle fibers and slowed skeletal muscle regeneration. In conclusion, these data provide preliminary insights into the biological functions of RRM2 in chicken muscle development and skeletal muscle regeneration.

Comparative analyses of dynamic transcriptome profiles highlight key response genes and dominant isoforms for muscle development and growth in chicken

BackgroundModern breeding strategies have resulted in significant differences in muscle mass between indigenous chicken and specialized broiler. However, the molecular regulatory mechanisms that underlie these differences remain elusive. The aim of this study was to identify key genes and regulatory mechanisms underlying differences in breast muscle development between indigenous chicken and specialized broiler.ResultsTwo time-series RNA-sequencing profiles of breast muscles were generated from commercial Arbor Acres (AA) broiler (fast-growing) and Chinese indigenous Lushi blue-shelled-egg (LS) chicken (slow-growing) at embryonic days 10, 14, and 18, and post-hatching day 1 and weeks 1, 3, and 5. Principal component analysis of the transcriptome profiles showed that the top four principal components accounted for more than 80% of the total variance in each breed. The developmental axes between the AA and LS chicken overlapped at the embryonic stages but gradually separated at the adult stages. Integrative investigation of differentially-expressed transcripts contained in the top four principal components identified 44 genes that formed a molecular network associated with differences in breast muscle mass between the two breeds. In addition, alternative splicing analysis revealed that genes with multiple isoforms always had one dominant transcript that exhibited a significantly higher expression level than the others. Among the 44 genes, the TNFRSF6B gene, a mediator of signal transduction pathways and cell proliferation, harbored two alternative splicing isoforms, TNFRSF6B-X1 and TNFRSF6B-X2. TNFRSF6B-X1 was the dominant isoform in both breeds before the age of one week. A switching event of the dominant isoform occurred at one week of age, resulting in TNFRSF6B-X2 being the dominant isoform in AA broiler, whereas TNFRSF6B-X1 remained the dominant isoform in LS chicken. Gain-of-function assays demonstrated that both isoforms promoted the proliferation of chicken primary myoblasts, but only TNFRSF6B-X2 augmented the differentiation and intracellular protein content of chicken primary myoblasts.ConclusionsFor the first time, we identified several key genes and dominant isoforms that may be responsible for differences in muscle mass between slow-growing indigenous chicken and fast-growing commercial broiler. These findings provide new insights into the regulatory mechanisms underlying breast muscle development in chicken.

Transcriptome analysis of mRNAs, lncRNAs, and miRNAs in the skeletal muscle of Tibetan chickens at different developmental stages.

Introduction: As a valuable genetic resource, native birds can contribute to the sustainable development of animal production. Tibetan chickens, known for their special flavor, are one of the important local poultry breeds in the Qinghai-Tibet Plateau. However, Tibetan chickens have a slow growth rate and poor carcass traits compared with broilers. Although most of the research on Tibetan chickens focused on their hypoxic adaptation, there were fewer studies related to skeletal muscle development. Methods: Here, we performed the transcriptional sequencing of leg muscles from Tibetan chicken embryos at E (embryonic)10, E14, and E18. Results: In total, 1,600, 4,610, and 2,166 DE (differentially expressed) mRNAs, 210, 573, and 234 DE lncRNAs (long non-coding RNAs), and 52, 137, and 33 DE miRNAs (microRNAs) were detected between E10 and E14, E10 and E18, and E14 and E18, respectively. Functional prediction showed several DE mRNAs and the target mRNAs of DE lncRNAs and DE miRNAs were significantly enriched in sarcomere organization, actin cytoskeleton organization, myofibril, muscle fiber development, and other terms and pathways related to muscle growth and development. Finally, a lncRNA-miRNA-mRNA ceRNA (competing endogenous RNA) network associated with muscle growth and development, which contained 6 DE lncRNAs, 13 DE miRNAs, and 50 DE mRNAs, was constructed based on the screened DE RNAs by Gene Ontology (GO) enrichment. These DE RNAs may play a critical regulatory role in the skeletal muscle development of chickens. Discussion: The results provide a genomic resource for mRNAs, lncRNAs, and miRNAs potentially involved in the skeletal muscle development of chickens, which lay the foundation for further studies of the molecular mechanisms underlying skeletal muscle growth and development in Tibetan chickens.