Chicken Skeletal Muscle Development Research Articles

MiR-460b-5p promotes proliferation and differentiation of chicken myoblasts and targets RBM19 gene

The meat production of broilers is crucial to economic benefits of broiler industries, while the slaughter performance of broilers is directly determined by skeletal muscle development. Hence, the broiler breeding for growth traits shows a great importance. As a kind of small noncoding RNA, microRNA (miRNA) can regulate the expression of multiple genes and perform a wide range of regulation in organisms. Currently, more and more studies have confirmed that miRNAs are closely associated with skeletal muscle development of chickens. Based on our previous miR-seq analysis (accession number: PRJNA668199), miR-460b-5p was screened as one of the key miRNAs probably involved in the growth regulation of chickens. However, the regulatory effect of miR-460b-5p on the development of chicken skeletal muscles is still unclear. Therefore, miR-460b-5p was further used for functional validation at the cellular level in this study. The expression pattern of miR-460b-5p was investigated in proliferation and differentiation stages of chicken primary myoblasts. It was showed that the expression level of miR-460b-5p gradually decreased from the proliferation stage (GM 50%) to the lowest at 24 h of differentiation. As differentiation proceeded, miR-460b-5p expression increased significantly, reaching the highest and stabilizing at 72 h and 96 h of differentiation. Through mRNA quantitative analysis of proliferation marker genes, CCK-8 and Edu assays, miR-460b-5p was found to significantly facilitate the transition of myoblasts from G1 to S phase and promote chicken myoblast proliferation. mRNA and protein quantitative analysis of differentiation marker genes, as well as the indirect immunofluorescence results of myotubes, revealed that miR-460b-5p significantly stimulated myotube development and promote chicken myoblast differentiation. In addition, the target relationship was validated for miR-460b-5p according to the dual-luciferase reporter assay and mRNA quantitative analysis, which indicates that miR-460b-5p was able to regulate RBM19 expression by specifically binding to the 3' UTR of RBM19. In summary, miR-460b-5p has positive regulatory effects on the proliferation and differentiation of chicken myoblasts, and RBM19 is a target gene of miR-460b-5p.

CircLRRFIP1 promotes the proliferation and differentiation of chicken skeletal muscle satellite cells by sponging the miR-15 family via activating AKT3-mTOR/p70S6K signaling pathway

Skeletal muscle is important for animal meat production, regulating movements, and maintaining homeostasis. Circular RNAs (circRNAs) have been founded to play vital role in myogenesis. However, the effects of the numerous circRNAs on growth and development of the skeletal muscle are yet to be uncovered. Herein, we identified circLRRFIP1, which is a novel circular RNA that is preferentially expressed in the skeletal muscle. To study the role of circLRRFIP1 in the skeletal muscle, the skeletal muscle satellite cells (SMSCs) was used to silenced or overexpressed circLRRFIP1. The results obtained in this study showed that circLRRFIP1 play a positive role in the proliferation and differentiation of SMSCs. The SMSCs were generated with stable knockdown and overexpression of circLRRFIP1, and the results showed that circLRRFIP1 exerts a stimulatory effect on the proliferation and differentiation of SMSCs. We further generated SMSCs with stable knockdown and overexpression of circLRRFIP1, and the results revealed that circLRRFIP1 exerts a stimulatory effect on the proliferation and differentiation of SMSCs. Mechanistically, circLRRFIP1 targets the myogenic inhibitory factor-miR-15 family to release the suppression of the miR-15 family to AKT3. The knockdown of AKT inhibits SMSC differentiation through the mTOR/p70S6K pathway. Taken together, the results obtained in this present study revealed the important role and the regulatory mechanisms of circLRRFIP1 in the development of chicken skeletal muscle. Therefore, this study provides an attractive target for molecular breeding to enhance meat production in the chicken industry.

Transcriptome-Based Identification of the Muscle Tissue-Specific Expression Gene CKM and Its Regulation of Proliferation, Apoptosis and Differentiation in Chicken Primary Myoblasts.

Skeletal muscle is an essential tissue in meat-producing animals, and meat-producing traits have been a hot topic in chicken genetic breeding research. Current research shows that creatine kinase M-type-like (CKM) is one of the most abundant proteins in skeletal muscle and plays an important role in the growth and development of skeletal muscle, but its role in the development of chicken skeletal muscle is still unclear. Via RNA sequencing (RNA-seq), we found that CKM was highly expressed in chicken breast muscle tissue. In this study, the expression profile of CKM was examined by quantitative real-time PCR (qPCR), and overexpression and RNA interference techniques were used to explore the functions of CKM in the proliferation, apoptosis and differentiation of chicken primary myoblasts (CPMs). It was shown that CKM was specifically highly expressed in breast muscle and leg muscle and was highly expressed in stage 16 embryonic muscle, while CKM inhibited proliferation, promoted the apoptosis and differentiation of CPMs and was involved in regulating chicken myogenesis. Transcriptome sequencing was used to identify genes that were differentially expressed in CPMs after CKM disruption, and bioinformatics analysis showed that CKM was involved in regulating chicken myogenesis. In summary, CKM plays an important role in skeletal muscle development during chicken growth and development.

The VGLL2 gene participates in muscle development in Gushi chickens1

Previous studies have shown that VGLL2, a member of the VGLL family, plays important roles in the growth and development of animal skeletal muscle, but its specific role in the development of chicken skeletal muscle is unclear. The main goal of this study was to explore the biological functions of VGLL2 in the development of chicken skeletal muscle and the proliferation and differentiation of skeletal muscle cells in vitro. In this study, we detected the effect of VGLL2 on the proliferation of myoblasts by CCK8, EdU and flow cytometry analyses after overexpressing and interfering with VGLL2. Indirect immunofluorescence was used to detect the effect of VGLL2 on the differentiation of myoblasts. qRT-PCR and hematoxylin and eosin (H&E) staining were used to evaluate the effects of VGLL2 overexpression on the growth rate and muscle fiber structure of chicken skeletal muscle. The results showed that VGLL2 inhibited the proliferation of primary cultured chicken myoblasts and promoted the differentiation of these cells. Interestingly, food intake and muscle fiber development were significantly enhanced by the overexpression of VGLL2 in chickens. Taken together, these data demonstrate that the VGLL2 gene may be a useful marker for improving muscle mass in poultry.

Gga-miRNA-181-5p family facilitates chicken myogenesis via targeting TGFBR1 to block TGF-β signaling

MicroRNAs (miRNAs) have been demonstrated to control chicken skeletal muscle growth, however, the potential function of the miR-181-5p family in chicken myogenesis remains largely unknown. Here, our study identified the two chicken (Gallus gallus; Gga) miR-181-5p family members widely expressed in various tissues, specifically miR-181a-5p and miR-181b-5p. Besides, the breast muscles of fast-growing broilers expressed higher levels of miR-181a-5p and miR-181b-5p than those of slow-growing layers. Functionally, miR-181a-5p and miR-181b-5p both promote the expression level of myogenic factors including Myogenin (MyoG), Myogenic differentiation 1 (MyoD1), and Myosin heavy chain (MyHC), meanwhile accelerating the myotube formation of skeletal muscle satellite cells (SMSCs). Mechanistically, miR-181a-5p and miR-181b-5p directly bind to the 3’ Untranslated region (UTR) of the Transforming growth factor beta receptor 1 (TGFBR1) mRNA, further reducing the expression of TGFBR1. TGFBR1 is a key Transforming growth factor beta (TGF-β) signaling transduction receptor and had a negative function in muscle cell differentiation. Furthermore, knockdown of TGFBR1 facilitated the expression of chicken myogenic factors, boosted myotube formation, and decreased the SMAD family member 2/3 (SMAD2/3) phosphorylation in chicken SMSCs. SMAD2/3 are downstream of TGF-β signaling, and miR-181a-5p and miR-181b-5p could reduce the expression of TGFBR1 to further diminish the SMAD2/3 phosphorylation. Our findings revealed that the miR-181-5p family targets TGFBR1 to break the TGF-β signaling transduction, which resulted in promoting chicken skeletal muscle development.

CircRNAs Related to Breast Muscle Development and Their Interaction Regulatory Network in Gushi Chicken.

Circular RNAs (circRNAs) play a significant regulatory role during skeletal muscle development. To identify circRNAs during postnatal skeletal muscle development in chickens, we constructed 12 cDNA libraries from breast muscle tissues of Chinese Gushi chickens at 6, 14, 22, and 30 weeks and performed RNA sequencing. In total, 2112 circRNAs were identified, and among them 79.92% were derived from exons. CircRNAs are distributed on all chromosomes of chickens, especially chromosomes 1-9 and Z. Bioinformatics analysis showed that each circRNA had an average of 38 miRNA binding sites, 61.32% of which have internal ribosomal entry site (IRES) elements. Furthermore, in total 543 differentially expressed circRNAs (DE-circRNAs) were identified. Functional enrichment analysis revealed that DE-circRNAs source genes are engaged in biological processes and muscle development-related pathways; for example, cell differentiation, sarcomere, and myofibril formation, mTOR signaling pathway, and TGF-β signaling pathway, etc. We also established a competitive endogenous RNA (ceRNA) regulatory network associated with skeletal muscle development. The results in this report indicate that circRNAs can mediate the development of chicken skeletal muscle by means of a complex ceRNA network among circRNAs, miRNAs, genes, and pathways. The findings of this study might help increase the number of known circRNAs in skeletal muscle tissue and offer a worthwhile resource to further investigate the function of circRNAs in chicken skeletal muscle development.

CircSUCO promotes proliferation and differentiation of chicken skeletal muscle satellite cells via sponging miR-15

ABSTRACT 1. In a previous high-throughput sequencing study, a novel circular RNA (circRNA) generated from a SUN domain containing ossification factor (SUCO) gene transcript (circSUCO) was differentially expressed during the embryonic muscle development. This study aimed to further explore the effect of circSUCO on chicken skeletal muscle development. 2. The experiment analysed the expression patterns of circSUCO in Tianfu broilers and clarified its function in the chicken skeletal muscle satellite cells (SMSC) after circSUCO knockdown. The qPCR results showed circSUCO was highly expressed in skeletal muscle and has different expression levels during various development periods. 3. Mechanistically, a series of in vitro experiments showed that circSUCO interference suppressed proliferation and differentiation of SMSC. In addition, it was observed that circSUCO competitively binds with microRNAs such as miR-15a, miR-15b-5p, and miR-15c-5p according to the dual-luciferase assay and qPCR. 4. Correlation was positive between the circSUCO expression level and the ratio of the breast muscle. The results revealed that circSUCO could play a positive role in proliferation and differentiation of SMSC via sponging miR-15a, miR-15b-5p, and miR-15c-5p, hence, may contribute to skeletal muscle development in chicken.

MyoG-enhanced circGPD2 regulates chicken skeletal muscle development by targeting miR-203a

Circular RNAs (circRNAs) are a subclass of RNA macromolecules that are reported to be involved in the regulation of skeletal muscle development. However, the functions and regulatory mechanisms of circRNAs in chicken myogenesis are still largely unclear. Here, we identified a novel circRNA, circGPD2, an RNA macromolecule with a calculated molecular weight of 215 kDa. We discovered that circGPD2 is a muscle-specific circRNA and is strongly expressed in the breast muscle of broilers by utilizing the comparison model of layers and broilers. Functional analysis revealed circGPD2 has a positive role in the proliferation and differentiation of myoblasts, and circGPD2 performs function through the release of the inhibition effect of miR-203a on c-JUN and MEF2C. Besides, the myogenic regulatory factor MyoG enhanced the expression of circGPD2 by targeting the E-box element on the GPD2 promoter. Importantly, lentivirus-mediated circGPD2 knockdown resulted in the breast muscle mass loss of the chicks. Overall, we revealed the crucial role of circGPD2 in chicken myogenesis in vitro and in vivo, and analyzed the upstream and downstream regulation mechanisms of circGPD2. Our study provides an attractive target for molecular marker-assisted breeding to improve the meat yield in the chicken meat industry.

The expression, function, and coding potential of circular RNA circEDC3 in chicken skeletal muscle development

As an emerging class of non-coding transcripts, circular RNAs (circRNAs) are proved to participate in the complex process of myogenesis in diverse species. A previous study has identified circular RNA EDC3 (circEDC3) as a typical covalently closed circular RNA abundant in chicken skeletal muscle. This study found that circEDC3 is a conservative circular RNA and performed functional analysis to investigate the role of circEDC3 in chicken muscle growth. The results indicated that circEDC3 could inhibit (P<0.05) chicken skeletal muscle satellite cells (SMSCs) proliferation and differentiation but had no significant influence on SMSCs apoptosis. Additionally, bioinformatics analysis showed that circEDC3 had promising coding potential. The open reading frames (ORF) were found in circEDC3 in this study. Furthermore, this study predicted that circEDC3 had internal ribosome entry sites (IRES) and N6-methyladenosine (m6A) motifs in different species, implying that circEDC3 might be translatable. This study revealed that circEDC3 might be a negative regulator in chicken muscle development and suggested it has protein-coding potential in different species.

CircCCDC91 regulates chicken skeletal muscle development by sponging miR-15 family via activating IGF1-PI3K/AKT signaling pathway

Circular RNAs (circRNAs) has been reported in various tissues of animals and associated with multiple biological processes. From our previous sequencing data, we found a novel circRNA, circCCDC91 which was generated from exon 2 to 8 of the CCDC91 gene. We observed that circCCDC91 was differentially expressed in the chicken breast muscle among 4 different embryonic developmental time points (embryonic day 10 [E10], E13, E16, and E19). Therefore, we assumed that circCCDC91 have a potential function in chicken skeletal muscle development. In this study, we firstly verify the annular structure and expression pattern of circCCDC91, and further investigate on whether circCCDC91 could promote chicken skeletal development. Mechanistically, circCCDC91 could absorb miR-15a, miR-15b-5p, and miR-15c-5p to modulate the expression of Insulin receptor substrate1 (IRS1), as well as activate insulin-1ike growth factor 1-phosphatidylinositol 3-kinase/AKT (IGF1-PI3K/AKT) signaling pathway. In addition, circCCDC91 could rescue skeletal muscle atrophy by activating IGF1-PI3K/AKT pathway. Taken together, the findings in this study revealed that the newly identified circCCDC91 promotes myoblasts proliferation and differentiation, and alleviates skeletal muscle atrophy by directly binding to miR-15 family via activating IGF1-PI3K/AKT signaling pathway in chicken.

Circular RNA circFNDC3AL Upregulates BCL9 Expression to Promote Chicken Skeletal Muscle Satellite Cells Proliferation and Differentiation by Binding to miR-204.

Skeletal muscle is a heterogeneous tissue that is essential for initiating movement and maintaining homeostasis. The genesis of skeletal muscle is an integrative process that lasts from embryonic development to postnatal stages, which is carried out under the modulation of many factors. Recent studies have shown that circular RNAs (circRNAs), a class of non-coding RNAs, are involved in myogenesis. However, more circRNAs and their mechanisms that may regulate skeletal muscle development remain to be explored. Through in-depth analysis of our previous RNA-Seq data, circFNDC3AL was found to be a potentially functional circRNA highly expressed during embryonic development of chicken skeletal muscle. Therefore, in this study, we investigated the effect of circFNDC3AL on skeletal muscle development in chickens and found that circFNDC3AL promoted chicken skeletal muscle satellite cell (SMSC) proliferation and differentiation. To gain a thorough understanding of the exact modulatory mechanisms of circFNDC3AL in chicken skeletal muscle development, we performed target miRNA analysis of circFNDC3AL and found that circFNDC3AL has a binding site for miR-204. Subsequently, we demonstrated that miR-204 inhibited chicken SMSC proliferation and differentiation, which showed the opposite functions of circFNDC3AL. Furthermore, we identified the miR-204 target gene B-cell CLL/lymphoma 9 (BCL9) and validated that miR-204 had an inhibitory effect on BCL9, while the negative effect could be relieved by circFNDC3AL. In addition, we verified that BCL9 performed the same positive functions on chicken SMSC proliferation and differentiation as circFNDC3AL, as opposed to miR-204. In conclusion, our study identified a circRNA circFNDC3AL that upregulates BCL9 expression to promote the proliferation and differentiation of chicken SMSCs by binding to miR-204.

PDLIM5 Affects Chicken Skeletal Muscle Satellite Cell Proliferation and Differentiation via the p38-MAPK Pathway.

Simple SummaryPDZ and LIM domain 5 (PDLIM5) can increase C2C12 cell differentiation; however, the role of PDLIM5 in chicken skeletal muscle satellite cells (SMSCs) is unclear. In this study, the effect of PDLIM5 was verified on SMSCs in vitro, and then the molecular mechanism was determined by transcriptome sequencing. We demonstrated that PDLIM5 can positively affect chicken SMSC proliferation and differentiation via the p38-MAPK (mitogen activated kinase-like protein) pathway. These results indicate that PDLIM5 may be involved in chicken skeletal muscle growth and development.Skeletal muscle satellite cell growth and development is a complicated process driven by multiple genes. The PDZ and LIM domain 5 (PDLIM5) gene has been proven to function in C2C12 myoblast differentiation and is involved in the regulation of skeletal muscle development. The role of PDLIM5 in chicken skeletal muscle satellite cells, however, is unclear. In this study, in order to determine whether the PDLIM5 gene has a function in chicken skeletal muscle satellite cells, we examined the changes in proliferation and differentiation of chicken skeletal muscle satellite cells (SMSCs) after interfering and overexpressing PDLIM5 in cells. In addition, the molecular pathways of the PDLIM5 gene regulating SMSC proliferation and differentiation were analyzed by transcriptome sequencing. Our results show that PDLIM5 can promote the proliferation and differentiation of SMSCs; furthermore, through transcriptome sequencing, it can be found that the differential genes are enriched in the MAPK signaling pathway after knocking down PDLIM5. Finally, it was verified that PDLIM5 played an active role in the proliferation and differentiation of chicken SMSCs by activating the p38-MAPK signaling pathway. These results indicate that PDLIM5 may be involved in the growth and development of chicken skeletal muscle.

Identification of diverse cell populations in skeletal muscles and biomarkers for intramuscular fat of chicken by single-cell RNA sequencing

BackgroundThe development of skeletal muscle is closely related to the efficiency of meat production and meat quality. Chicken skeletal muscle development depends on myogenesis and adipogenesis and occurs in two phases—hyperplasia and hypertrophy. However, cell profiles corresponding to the two-phase muscle development have yet to be determined. Single-cell RNA-sequencing (scRNA-seq) can elucidate the cell subpopulations in tissue and capture the gene expression of individual cells, which can provide new insights into the myogenesis and intramuscular adipogenesis.ResultsTen cell clusters at the post-hatching developmental stage at Day 5 and seven cell clusters at the late developmental stage at Day 100 were identified in chicken breast muscles by scRNA-seq. Five myocyte-related clusters and two adipocyte clusters were identified at Day 5, and one myocyte cluster and one adipocyte cluster were identified at Day 100. The pattern of cell clustering varied between the two stages. The cell clusters showed clear boundaries at the terminal differentiation stage at Day 100; by contrast, cell differentiation was not complete at Day 5. APOA1 and COL1A1 were selected from up-regulated genes in the adipocyte cluster and found to be co-expressed with the ADIPOQ adipocyte marker gene in breast muscles by RNA in situ hybridization.ConclusionsThis study is the first to describe the heterogeneity of chicken skeletal muscle at two developmental stages. The genes APOA1 and COL1A1 were identified as biomarkers for chicken intramuscular fat cells.

The effect of two different green lighting schedules during embryogenesis on myogenesis in broiler chickens

Abstract Light is one of the factors that interfere with avian development as early as during embryogenesis. The aim of this study was to determine the effect of monochromatic green light on hatchability, body weight and breast muscle myogenesis in broiler chickens, as expressed by breast muscle weight, muscle fibre thickness and number of satellite cells (Pax7 +) on d 1, 4 and 8 of life. Lighting programmes investigated were two different light cycles of 30 minutes of light and 30 minutes of darkness (L1 30/30), 12Â h of light and 12Â h of darkness (L2 12/12) and one control group (C) of continuous darkness applied throughout embryogenesis until day 18 of incubation. Green light was found to have a positive effect, resulting in significantly greater body weight of the exposed chickens (group: L2 12/12 – on d 4 of age; group L1 30/30 – on d 8 of age), and highly significantly greater fibre thickness in breast muscle in group L1 on d 8 of age. Photo-stimulation exerted a significant effect on the number of Pax7 + cells per unit of muscle cross-sectional area in broiler chickens on d 4 of life. However, higher body weight tended to be accompanied by a decrease in the number of Pax7 + satellite cells, calculated per unit of muscle area. This indicates the fusion of satellite cells with existing muscle fibres, which leads to an increase in their diameter. Based on the obtained results, we conclude that there is a potential opportunity to regulate the growth and development of chicken skeletal muscle by light during embryogenesis. This may further increase the effectiveness of chicken meat production. However, more research is needed in order to find the most optimal lighting treatment.

Analyses of MicroRNA and mRNA Expression Profiles Reveal the Crucial Interaction Networks and Pathways for Regulation of Chicken Breast Muscle Development.

There is a lack of understanding surrounding the molecular mechanisms involved in the development of chicken skeletal muscle in the late postnatal stage, especially in the regulation of breast muscle development related genes, pathways, miRNAs and other factors. In this study, 12 cDNA libraries and 4 small RNA libraries were constructed from Gushi chicken breast muscle samples from 6, 14, 22, and 30 weeks. A total of 15,508 known transcripts, 25,718 novel transcripts, 388 known miRNAs and 31 novel miRNAs were identified by RNA-seq in breast muscle at the four developmental stages. Through correlation analysis of miRNA and mRNA expression profiles, it was found that 417, 370, 240, 1,418, 496, and 363 negatively correlated miRNA–mRNA pairs of W14 vs. W6, W22 vs. W6, W22 vs. W14, W30 vs. W6, W30 vs. W14, and W30 vs. W22 comparisons, respectively. Based on the annotation analysis of these miRNA–mRNA pairs, we constructed the miRNA–mRNA interaction network related to biological processes, such as muscle cell differentiation, striated muscle tissue development and skeletal muscle cell differentiation. The interaction networks for signaling pathways related to five KEGG pathways (the focal adhesion, ECM-receptor interaction, FoxO signaling, cell cycle, and p53 signaling pathways) and PPI networks were also constructed. We found that ANKRD1, EYA2, JSC, AGT, MYBPC3, MYH11, ACTC1, FHL2, RCAN1, FOS, EGR1, and FOXO3, PTEN, AKT1, GADD45, PLK1, CCNB2, CCNB3 and other genes were the key core nodes of these networks, most of which are targets of miRNAs. The FoxO signaling pathway was in the center of the five pathway-related networks. In the PPI network, there was a clear interaction among PLK1 and CDK1, CCNB2, CDK1, and GADD45B, and CDC45, ORC1 and MCM3 genes. These results increase the understanding for the molecular mechanisms of chicken breast muscle development, and also provide a basis for studying the interactions between genes and miRNAs, as well as the functions of the pathways involved in postnatal developmental regulation of chicken breast muscle.

Sequencing and characterization of lncRNAs in the breast muscle of Gushi and Arbor Acres chickens.

Chicken muscle quality is one of the most important factors determining the economic value of poultry, and muscle development and growth are affected by genetics, environment, and nutrition. However, little is known about the molecular regulatory mechanisms of long non-coding RNAs (lncRNAs) in chicken skeletal muscle development. Our study aimed to better understand muscle development in chickens and thereby improve meat quality. In this study, Ribo-Zero RNA-Seq was used to investigate differences in the expression profiles of muscle development related genes and associated pathways between Gushi (GS) and Arbor Acres (AA) chickens. We identified two muscle tissue specific expression lncRNAs. In addition, the target genes of these lncRNAs were significantly enriched in certain biological processes and molecular functions, as demonstrated by Gene Ontology (GO) analysis, and these target genes participate in five signaling pathway, as revealed by an analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Taken together, these data suggest that different lncRNAs might be involved in regulating chicken muscle development and growth and provide new insight into the molecular mechanisms of lncRNAs.

Copy Number Variation in SOX6 Contributes to Chicken Muscle Development.

Copy number variations (CNVs), which cover many functional genes, are associated with complex diseases, phenotypic diversity and traits that are economically important to raising chickens. The sex-determining region Y-box 6 (Sox6) plays a key role in fast-twitch muscle fiber differentiation of zebrafish and mice, but it is still unknown whether SOX6 plays a role in chicken skeletal muscle development. We identified two copy number polymorphisms (CNPs) which were significantly related to different traits on the genome level in chickens by AccuCopy® and CNVplex® analyses. Notably, five white recessive rock (CN = 1, CN = 3) variant individuals and two Xinghua (CN = 3) variant individuals contain a CNP13 (chromosome5: 10,500,294–10,675,531) which overlaps with SOX6. There is a disordered region in SOX6 proteins 265–579 aa coded by a partial CNV overlapping region. A quantitative real-time polymerase chain reaction showed that the expression level of SOX6 mRNA was positively associated with CNV and highly expressed during the skeletal muscle cell differentiation in chickens. After the knockdown of the SOX6, the expression levels of IGFIR1, MYF6, SOX9, SHOX and CCND1 were significantly down-regulated. All of them directly linked to muscle development. These results suggest that the number of CNVs in the CNP13 is positively associated with the expression level of SOX6, which promotes the proliferation and differentiation of skeletal muscle cells by up-regulating the expression levels of the muscle-growth-related genes in chickens as in other animal species.

Ebp1 regulates myogenic differentiation of myoblast cells via SMAD2/3 signaling pathway.

Regulation of skeletal muscle development requires many of the regulatory networks that are fundamental to developmental myogenesis. ErbB3 binding protein-1 (Ebp1) is involved in the control of myoblasts development in chicken. However, the expression and biological functions of Ebp1 in the progress of myogenesis are unclear. This study focused on determining the effect of Ebp1 on myogenic proliferation and differentiation using a primary myoblasts culture model. Ebp1 was found to upregulate in proliferating myoblasts and decrease at the early stage of myogenic differentiation. The level of endogenous Ebp1 increased from E9 to E20 chicken leg muscles. Knockdown of Ebp1 had no effect on myoblasts proliferation. However, myogenic differentiation into multinucleated myotubes was significantly reduced. The mRNA and protein expression of MRFs was decreased when Ebp1 was knocked down. Downregulation of Ebp1, accompanied by elevated levels of pSMAD2/3, suggests that Ebp1 is involved in regulating myogenic differentiation via SMAD2/3 inhibition. The phosphorylation of SMAD2/3 was activated and the expression of MYOD and MYOG was reduced in Ebp1 knockdown myoblasts, but addition of LY2109761 (an inhibitor specified to SMAD2/3) blocked these effects. Collectively, these results indicate that Ebp1 promotes myoblast differentiation by inhibition of SMAD2/3 signaling pathway during chicken myogenesis. These data provide new insights into the biological role of Ebp1 in embryonic chicken skeletal muscle development.

Differentiation of expression profiles of two calcineurin subunit genes in chicken skeletal muscles during early postnatal growth depending on anatomical location of muscles and breed

Calcineurin (Cn or CaN) is implicated in the control of skeletal muscle fiber phenotype and hypertrophy. However, little information is available concerning the expression of Cn in chickens. In the present study, the expression of two Cn subunit genes (CnAα and CnB1) was quantified by qPCR in the lateral gastrocnemius (LG, mainly composing of red fast-twitch myofibers), the soleus (mainly composing of red slow-twitch myofibers) and the extensor digitorum longus (EDL, mainly composing of white fast-twitch myofibers) from Qingyuan partridge chickens (QY, slow-growing chicken breed) and Recessive White chickens (RW, fast-growing chicken breed) on different days (1, 8, 22, 36, 50 and 64 days post-hatching). Although CnAα and CnB1 gene expressions were variable with different trends in different skeletal muscles in the two chicken breeds during postnatal growth, it is highly muscle phenotype and breed specific. In general, the levels of CnAα and CnB1 gene expressions of the soleus were lower than those of EDL and LG in both chicken breeds at the same stages. Compared between the two chicken breeds, the levels of CnAα gene expression of the three skeletal muscles in QY chickens were higher than those in RW chickens on days 1 and 22. However, on day 64, the levels of both CnAα and CnB1 gene expressions of the three skeletal muscles were lower in QY chickens than those in RW chickens. Correlation analysis of the levels of CnAα and CnB1 gene expressions of the same skeletal muscle showed that there were positive correlations for all three skeletal muscle tissues in two chicken breeds. These results provide some valuable clues to understand the role of Cn in the development of chicken skeletal muscles, with a function that may be related to meat quality.

Integrative Analyses of miRNA-mRNA Interactions Reveal let-7b, miR-128 and MAPK Pathway Involvement in Muscle Mass Loss in Sex-Linked Dwarf Chickens.

The sex-linked dwarf (SLD) chicken is an ideal model system for understanding growth hormone (GH)-action and growth hormone receptor (GHR) function because of its recessive mutation in the GHR gene. Skeletal muscle mass is reduced in the SLD chicken with a smaller muscle fiber diameter. Our previous study has presented the mRNA and miRNA expression profiles of the SLD chicken and normal chicken between embryo day 14 and seven weeks of age. However, the molecular mechanism of GHR-deficient induced muscle mass loss is still unclear, and the key molecules and pathways underlying the GHR-deficient induced muscle mass loss also remain to be illustrated. Here, by functional network analysis of the differentially expressed miRNAs and mRNAs between the SLD and normal chickens, we revealed that let-7b, miR-128 and the MAPK pathway might play key roles in the GHR-deficient induced muscle mass loss, and that the reduced cell division and growth are potential cellular processes during the SLD chicken skeletal muscle development. Additionally, we also found some genes and miRNAs involved in chicken skeletal muscle development, through the MAPK, PI3K-Akt, Wnt and Insulin signaling pathways. This study provides new insights into the molecular mechanism underlying muscle mass loss in the SLD chickens, and some regulatory networks that are crucial for chicken skeletal muscle development.