Chicken Myoblasts Research Articles

Circular RNA circHIPK3 Promotes the Proliferation and Differentiation of Chicken Myoblast Cells by Sponging miR-30a-3p.

Circular RNAs and microRNAs widely exist in various species and play crucial roles in multiple biological processes. It is essential to study their roles in myogenesis. In our previous sequencing data, both miR-30a-3p and circular HIPK3 (circHIPK3) RNA, which are produced by the third exon of the HIPK3 gene, were differentially expressed among chicken skeletal muscles at 11 embryo age (E11), 16 embryo age (E16), and 1-day post-hatch (P1). Here, we investigated their potential roles in myogenesis. Proliferation experiment showed that miR-30a-3p could inhibit the proliferation of myoblast. Through dual-luciferase assay and Myosin heavy chain (MYHC) immunofluorescence, we found that miR-30a-3p could inhibit the differentiation of myoblast by binding to Myocyte Enhancer Factor 2 C (MEF2C), which could promote the differentiation of myoblast. Then, we found that circHIPK3 could act as a sponge of miR-30a-3p and exerted a counteractive effect of miR-30a-3p by promoting the proliferation and differentiation of myoblasts. Taking together, our data suggested that circHIPK3 could promote the chicken embryonic skeletal muscle development by sponging miR-30a-3p.

Gga-mir-133a-3p Regulates Myoblasts Proliferation and Differentiation by Targeting PRRX1.

Non-coding RNAs play a regulatory role in the growth and development of skeletal muscle. Our previous study suggested that gga-mir-133a-3p was a potential candidate for regulating myoblast proliferation and differentiation in skeletal muscle. The purpose of our study was to reveal the regulatory mechanism of gga-mir-133a-3p in the proliferation and differentiation of chicken myoblasts. Through the detection of cell proliferation activity, cell cycle progression and EdU, we found that gga-mir-133a-3p can significantly inhibit the proliferation of myoblasts. In the process of myogenic differentiation, gga-mir-133a-3p is up-regulated, while gga-mir-133a-3p can significantly promote the up-regulation of differentiation-related muscle-derived factors, indicating that gga-mir-133a-3p can promote the differentiation of myoblasts. Validation at the transcriptional level and protein level proved that gga-mir-133a-3p can inhibit the expression of PRRX1, and the dual-luciferase assay also showed their direct targeting relationship. Correspondingly, PRRX1 can significantly promote myoblast proliferation and inhibit myoblast differentiation. In our study, we confirmed that gga-mir-133a-3p participates in the regulation of proliferation and differentiation of myoblasts by targeting PRRX1.

Forkhead box O3 promotes cell proliferation and inhibits myotube differentiation in chicken myoblast cells

ABSTRACT1. In the poultry industry, growth performance is important due to its effects on economic value. Much effort has been put forth to achieve introgression of specific genes and DNA markers related to muscle proliferation and differentiation in selective breeding approaches.2. This study investigated the biological functions of the gene Forkhead box O3 (FOXO3) during myogenic differentiation in chicken myoblast cells. FOXO3 was downregulated in primary chicken myoblast (pCM) cells by the piggyBac transposon-mediated microRNA (miRNA) knock-down (KD) system.3. The pCM cells that were stably integrated into the FOXO3 KD expression vector showed significant downregulation of FOXO3 protein and mRNA levels. Expression levels of paired box protein Pax7 (Pax7) and target genes such as CCAAT/enhancer binding protein beta and serum response element decreased in FOXO3 KD pCM cells. In addition, in the undifferentiated myoblast stage, there were no significant differences in cell morphology; however, proliferation rate in FOXO3 KD pCM cells was significantly lower during d 4 and 5 of in vitro culture. By contrast, when myotube differentiation was induced, FOXO3 KD pCM cells exhibited rapid initiation of myotube formation, higher expression of myogenin and desmin as myogenic indicators and a further differentiated phenotype than observed in regular pCM cells.4. These results demonstrated that FOXO3 promotes cell proliferation and inhibits myotube differentiation in chicken myoblast cells. Therefore, the regulation of FOXO3 could be applied to improve muscle differentiation in commercial poultry.

Dissection of Myogenic Differentiation Signatures in Chickens by RNA-Seq Analysis.

A series of elaborately regulated and orchestrated changes in gene expression profiles leads to muscle growth and development. In this study, RNA sequencing was used to profile embryonic chicken myoblasts and fused myotube transcriptomes, long non-coding RNAs (lncRNAs), and messenger RNAs (mRNAs) at four stages of myoblast differentiation. Of a total of 2484 lncRNA transcripts, 2288 were long intergenic non-coding RNAs (lincRNAs) and 198 were antisense lncRNAs. Additionally, 1530 lncRNAs were neighboring 2041 protein-coding genes (<10 kb upstream and downstream) and functionally enriched in several pathways related to skeletal muscle development that have been extensively studied, indicating that these genes may be in cis-regulatory relationships. In addition, Pearson’s correlation coefficients demonstrated that 990 lncRNAs and 7436 mRNAs were possibly in trans-regulatory relationships. These co-expressed mRNAs were enriched in various developmentally-related biological processes, such as myocyte proliferation and differentiation, myoblast differentiation, and myoblast fusion. The number of transcripts (906 lncRNAs and 4422 mRNAs) differentially expressed across various stages declined with the progression of differentiation. Then, 4422 differentially expressed genes were assigned to four clusters according to K-means analysis. Genes in the K1 cluster likely play important roles in myoblast proliferation and those in the K4 cluster were likely associated with the initiation of myoblast differentiation, while genes in the K2 and K3 clusters were likely related to myoblast fusion. This study provides a catalog of chicken lncRNAs and mRNAs for further experimental investigations and facilitates a better understanding of skeletal muscle development.

Gga-let-7f-3p promotes apoptosis in selenium deficiency-induced skeletal muscle by targeting selenoprotein K.

Selenoprotein K (SELENOK) is primarily observed in the endoplasmic reticulum, and serves to maintain the normal physiological functions of skeletal muscle. Skeletal muscle development and regeneration are associated with significant changes in the expression of specific microRNAs (miRNAs). Downregulated SELENOK expression is observed in chicken muscles deficient of Se. However, the mechanisms of miRNA regulation of SELENOK expression remain elusive. Here, deep sequencing was used to detect the miRNA profiles of muscle in Se deficient (-Se group) and normal (C group) chickens. A dual-luciferase reporter assay was adopted to verify the relationship between SELENOK and gga-let-7f-3p. In addition, gga-let-7f-3p was either overexpressed or knocked-down in chicken myoblasts. Furthermore, the cells were treated with N-acetyl-l-cysteine (NAC) or hydrogen peroxide (H2O2) in order to probe the factors involved in oxidative stress, endoplasmic reticulum stress (ERS) and apoptosis, respectively. Relative to the C group, there were 132 differentially expressed miRNAs (including 57 upregulated and 75 downregulated) in the muscles of the -Se group. The dual-luciferase reporter assay showed that SELENOK was a primary target of gga-let-7f-3p. It was also observed that the overexpression or knock-down of gga-let-7f-3p significantly influenced the SELENOK expression. Moreover, NAC blocked mimics of ga-let-7f-3p, thus inducing oxidative stress, ERS and apoptosis. Simultaneously, gga-let-7f-3p inhibitors blocked the stimulant effects caused by H2O2 in chicken myoblasts. Furthermore, Se deficiency downregulated the SELENOK protein expression and induced oxidative stress, ERS and apoptosis in chicken muscles. In conclusion, the gga-let-7f-3p-SELENOK pathway played a pivotal role in Se deficiency mediated muscle injuries through the induction of oxidative stress and ERS, ultimately promoting apoptosis.

Transcriptomic analysis of chicken Myozenin 3 regulation reveals its potential role in cell proliferation.

Embryonic muscle development and fibre type differentiation has always been a topic of great importance due to its impact on both human health and farm animal financial values. Myozenin3 (Myoz3) is an important candidate gene that may regulate these processes. In the current study, we knocked down and overexpressed Myoz3 in chicken embryonic fibroblasts (CEFs) and chicken myoblasts, then utilized RNA-seq technology to screen genes, pathways and biological processes associated with Myoz3. Multiple differentially expressed genes were identified, including MYH10, MYLK2, NFAM1, MYL4, MYL9, PDZLIM1; those can in turn regulate each other and influence the development of muscle fibres. Gene ontology (GO) terms including some involved in positive regulation of cell proliferation were enriched. We further validated our results by testing the activity of cells by cell counting kit-8(CCK-8) and confirmed that under the condition of Myoz3 overexpression, the proliferation rate of CEFs and myoblasts was significantly upregulated, in addition, expression level of fast muscle specific gene was also significantly upregulated in myoblasts. Pathway enrichment analysis revealed that the PPAR (Peroxisome Proliferator-Activated Receptor) pathway was enriched, suggesting the possibility that Myoz3 regulates muscle fibre development and differentiation through the PPAR pathway. Our results provide valuable evidence regarding the regulatory functions of Myoz3 in embryonic cells by screening multiple candidate genes, biological processes and pathways associated with Myoz3.

Let-7b Regulates Myoblast Proliferation by Inhibiting IGF2BP3 Expression in Dwarf and Normal Chicken.

The sex-linked dwarf chicken is caused by the mutation of growth hormone receptor (GHR) gene and characterized by shorter shanks, lower body weight, smaller muscle fiber diameter and fewer muscle fiber number. However, the precise regulatory pathways that lead to the inhibition of skeletal muscle growth in dwarf chickens still remain unclear. Here we found a let-7b mediated pathway might play important role in the regulation of dwarf chicken skeletal muscle growth. Let-7b has higher expression in the skeletal muscle of dwarf chicken than in normal chicken, and the expression of insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), which is a translational activator of IGF2, showed opposite expression trend to let-7b. In vitro cellular assays validated that let-7b directly inhibits IGF2BP3 expression through binding to its 3′UTR region, and the protein level but not mRNA level of IGF2 would be reduced in let-7b overexpressed chicken myoblast. Let-7b can inhibit cell proliferation and induce cell cycle arrest in chicken myoblast through let-7b-IGF2BP3-IGF2 signaling pathway. Additionally, let-7b can also regulate skeletal muscle growth through let-7b-GHR-GHR downstream genes pathway, but this pathway is non-existent in dwarf chicken because of the deletion mutation of GHR 3′UTR. Notably, as the loss binding site of GHR for let-7b, let-7b has enhanced its binding and inhibition on IGF2BP3 in dwarf myoblast, suggesting that the miRNA can balance its inhibiting effect through dynamic regulate its binding to target genes. Collectively, these results not only indicate that let-7b can inhibit skeletal muscle growth through let-7b-IGF2BP3-IGF2 signaling pathway, but also show that let-7b regulates myoblast proliferation by inhibiting IGF2BP3 expression in dwarf and normal chickens.

Beta-carotene preferentially regulates chicken myoblast proliferation withdrawal and differentiation commitment via BCO1 activity and retinoic acid production

The enzyme β-carotene oxygenase 1 (BCO1) catalyzes the breakdown of provitamin A, including beta-carotene (BC), into retinal, prior to its oxidation into retinoic acid (RA). Allelic variation at the BCO1 locus results in differential expression of its mRNA and affects carotenoid metabolism specifically in chicken Pectoralis major muscle. In this context, the aim of this study was to evaluate the potential myogenic effect of BC and the underlying mechanisms in chicken myoblasts. BCO1 mRNA was detected in myoblasts derived from chicken satellite cells. Treating these myoblasts with BC led to a significant decrease in BrdU incorporation. This anti-proliferative effect was confirmed by a cell cycle study using flow cytometry. BC also significantly increased the differentiation index, suggesting a positive effect on the commitment of avian myoblasts to myogenic differentiation. Addition of DEAB, a specific inhibitor of RALDH activity, significantly reduced BC anti-proliferative and pro-differentiating effects, suggesting that BC exerted its biological effect on chicken myoblasts through activation of the RA pathway. We also observed that in myoblast showing decreased BCO1 expression consecutive to a natural mutation or to a siRNA treatment, the response to BC was inhibited. Nevertheless, BCO1 siRNA transfection increased expression of BCO2 which inhibited cell proliferation in control and BC treated cells.

Downregulated long noncoding RNA ALDBGALG0000005049 induces inflammation in chicken muscle suffered from selenium deficiency by regulating stearoyl-CoA desaturase.

Long non-coding RNAs (lncRNAs) have been demonstrated to play a pivotal role in proliferation and differentiation of muscles. However, the study on the roles of lncRNAs in Selenium (Se) deficiency induced muscle injury is still unclear. In this study, deep sequencing was performed to profile lncRNAs and mRNAs of the muscles from the Se deficiency (-Se group) and control (C group) chickens. The results revealed that 38 lncRNAs (23 up-regulated and 15 down-regulated) and 687 mRNAs (285 up-regulated and 402 down-regulated) were significantly dysregulated expressed, and the significantly dysregulated pathway including Phagosome, Cardiac muscle contraction, and Peroxisome Proliferator-Activated Receptor (PPAR) in -Se group. The regulatory relationship between ALDBGALG0000005049 and stearoyl-CoA desaturase (SCD), which involved in PPAR pathway was verified. The results also showed that the decreased expressions of SCD, PPARα, PPARβ and PPARγ, and the increased expressions of interleukin (IL)-1β, IL-6, IL-8, and chemokine (C-C motif) ligand 4 (CCL4) along with silencing of ALDBGALG0000005049 in chicken myoblasts. Moreover, increased expressions of IL-1β, IL-6, IL-8, and CCL4 and inflammatory cell infiltration in microstructure of chicken muscles treated with Se deficiency were observed. This study displayed an overview of aberrantly expressed lncRNAs and mRNAs profiles and PPAR pathway, and revealed that downregulation of ALDBGALG0000005049 caused inflammation by regulating SCD in chicken muscle resulted from Se deficiency.

Gene Silencing of Selenoprotein K Induces Inflammatory Response and Activates Heat Shock Proteins Expression in Chicken Myoblasts.

In the present study, specific small interfering RNA (siRNA) for selenoprotein K (Selk) gene was designed and transfected into chicken myoblasts. Then, the expressions of inflammatory factors (including induced nitric oxide synthase [iNOS], nuclear factor-kappa B [NF-κB], heme-oxygenase-1 [HO-1], cyclooxygenase-2 [COX-2], and prostaglandin E synthase [PTGEs]), inflammation-related cytokines (including interleukin [IL]-1β, IL-6, IL-7, IL-8, IL-17, and interferon [IFN]-γ), and heat shock proteins (HSPs) (including HSP27, HSP40, HSP60, HSP70, and HSP90) were examined at 24 and 72h after transfection. The results showed that messenger RNA (mRNA) expressions of iNOS, NF-κB, HO-1, COX-2, IL-6, IL-7, IL-8, HSP 27, HSP 40, HSP 60, HSP 70, and HSP 90 were significantly increased (p<0.05) at 24 and 72h after siRNA transfection, and the mRNA expressions of PTGEs, IL-1β, IL-17, and IFN-γ were significantly increased and decreased (p<0.05) at 24 and 72h after siRNA transfection. The results also showed that the protein expressions of iNOS, NF-κB, HO-1, COX-2, HSP60, HSP70, and HSP90 were significantly increased (p<0.05) at 24 and 72h after siRNA transfection. The correlation analysis and principal component analysis (PCA) showed that PTGEs, IL-1β, IL-17, IFN-γ, HSP40, and HSP90 might play special roles in response to Selk silencing in chicken myoblasts. These results indicated that Selk silencing induced inflammation response by affecting the expression levels of inflammatory factors and inflammation-related cytokines, and the heat shock proteins might play protective roles in this response in chicken myoblasts.

Gene expression and silencing of activin receptor type 2A (ACVR2A) in myoblast cells of chicken

Activin receptor type 2A (ACVR2A) acts as receptor for myostatin (MSTN) protein involved in inhibiting satellite cell proliferation and differentiation. The importance of the ACVR2A gene during embryonic and post-hatch periods in broiler and layer chicken was studied in an in vitro cell culture system.The expression pattern of the ACVR2A gene during embryonic stages was similar in broiler and layer lines. Post-hatch expression of the ACVR2A gene varied significantly between broiler and layer lines.Five shRNA molecules were designed to knockdown expression of the ACVR2A gene in chicken myoblast cells. The silencing of the ACVR2A gene in a cell culture system varied from 60% to 82%.It is concluded that between broiler and layer lines, there were no significant changes in expression of the ACVR2A gene during embryonic stages but it varied significantly during the post-hatch period. The shRNA showed silencing of the ACVR2A gene under an in vitro cell culture system.

Gene expression of selenoproteins can be regulated by selenoprotein K silencing in chicken myoblasts.

The aim of the present study was to clarify the effect of Selenoprotein K (Selk) silencing on the mRNA expression of 25 selenoproteins in chicken myoblasts. The specific small interfering RNA (siRNA) for Selk gene was designed and transfected into chicken myoblasts. Post-transfection mRNA expression of 25 selenoproteins was determined at various time periods i.e., 24, 48 and 72h. Moreover, based on the results of expression of 25 selenoproteins, correlation analysis and principal component analysis (PCA) were used for further analysis. The results showed that the designed siRNA effectively inhibited Selk expression (decreased by 20, 29 and 43% on 24, 48 and 72h, respectively) and the mRNA expression levels of the 23 selenoproteins were influenced by silencing Selk differently (P<0.05). Time-dependent pattern of mRNA expression after siRNA treatment in three groups were found similar: one group including Gpx1, Gpx2, Gpx3, Gpx4, Txnrd1, Txnrd2, Txnrd3, Sepw1, Selh, Sepp1, Selo and Sepx1, another group including Sepn1, Sels, Selt, Selm and Sep15 and other group including Dio2 and Dio3. The results of correlation analysis showed that Gpx1, Gpx2, Gpx3, Gpx4, Dio1, Dio3, Sepn1, Sels, Sepw1, Selt, Selh, Sep15, Seli and Selu had a positive correlation with Selk, while Dio2 and Sepp1 had a negative correlation with Selk. PCA data also indicated that Txnrd1, Txnrd2, Dio2, Selpb, Sepp1and Selo may play special roles in response to Selk silencing. In summary, these results indicated that different selenoproteins possess and exhibits distinct responses to silencing of Selk in chicken myoblasts.

Myomaker, Regulated by MYOD, MYOG and miR-140-3p, Promotes Chicken Myoblast Fusion.

The fusion of myoblasts is an important step during skeletal muscle differentiation. A recent study in mice found that a transmembrane protein called Myomaker, which is specifically expressed in muscle, is critical for myoblast fusion. However, the cellular mechanism of its roles and the regulatory mechanism of its expression remain unclear. Chicken not only plays an important role in meat production but is also an ideal model organism for muscle development research. Here, we report that Myomaker is also essential for chicken myoblast fusion. Forced expression of Myomaker in chicken primary myoblasts promotes myoblast fusion, whereas knockdown of Myomaker by siRNA inhibits myoblast fusion. MYOD and MYOG, which belong to the family of myogenic regulatory factors, can bind to a conserved E-box located proximal to the Myomaker transcription start site and induce Myomaker transcription. Additionally, miR-140-3p can inhibit Myomaker expression and myoblast fusion, at least in part, by binding to the 3ʹ UTR of Myomaker in vitro. These findings confirm the essential roles of Myomaker in avian myoblast fusion and show that MYOD, MYOG and miR-140-3p can regulate Myomaker expression.

Different responses of selenoproteins to the altered expression of selenoprotein W in chicken myoblasts

Selenoprotein W could influence certain selenoproteins expression through redox pathway.

Selenoprotein W serves as an antioxidant in chicken myoblasts

BackgroundSelenoprotein W (SelW) was thought to play an antioxidant role in mammals. Because chicken SelW has no cysteine (Cys) at the residue 37 (Cys37) that is required for the presumed antioxidant function in mammals, this study was conducted to determine whether chicken SelW possessed the same function. MethodsSmall interfering RNAs (siRNAs) technology was applied to suppress the SelW expression in chicken embryonic myoblasts. Thereafter, these myoblasts were treated with different concentrations of H2O2 and assayed for cell viability, apoptosis rate, reactive oxygen species (ROS) status, and expression levels of apoptosis-related genes and proteins (Bax, Bcl-2, and caspase-3). ResultsSilencing of the myoblast SelW gene decreased their cell viability, and increased their apoptosis rate and susceptibility to H2O2. While the knockout down of SelW up-regulated Bax and caspase-3 and down-regulated Bcl-2, the induced oxidative injuries were alleviated by treatment with a ROS scavenger, N-acetyl-l-cysteine (NAC). ConclusionChicken SelW protected embryonic myoblasts against cell apoptosis mediated by endogenous and exogenous H2O2. General significanceChicken SelW possesses antioxidant function similar to the mammalian homologues despite the lack of Cys37 in the peptide.

Possible Correlation between Selenoprotein W and Myogenic Regulatory Factors in Chicken Embryonic Myoblasts

The biological function of selenium (Se) is mainly elicited through Se-containing proteins. Selenoprotein W (SelW), one member of the selenoprotein family, is essential for the normal function of the skeletal muscle system. To investigate the possible relationship of Se in the process of differentiation in chicken myoblasts and the expression of SelW, the cultured chicken embryonic myoblasts were incubated with sodium selenite at different concentrations for 72h, and then the mRNA levels of SelW and myogenic regulatory factors (MRFs) in myoblasts were determined at 12, 24, 48, and 72h, respectively. Furthermore, the correlation between SelW mRNA expression and MRF mRNA expression was assessed. The results showed that the sodium selenite medium enhanced the mRNA expression of SelW, Myf-5, MRF4, and myogenin in chicken myoblasts. The mRNA expression levels of MRFs were significantly correlated with those of SelW at 24, 48, and 72h. These data demonstrate that Se is involved in the differentiation of chicken embryonic myoblasts, and SelW showed correlation with MRFs.

Administration of insulin to newly hatched chicks improves growth performance via impairment of MyoD gene expression and enhancement of cell proliferation in chicken myoblasts

The insulin/PI3K/Akt signaling pathway is strongly involved in the differentiation of C2C12 cells, as has been demonstrated by the addition of IGFs and insulin to culture media. In this study, we have characterized the role of insulin in chick myoblast proliferation and differentiation in vitro and in vivo, and have revealed novel details of how this exogenous hormone influences myogenic genes during differentiation. Chick myoblast cells cultured in differentiation medium (DMEM containing 2% FBS) supplemented with insulin exhibited a significant decrease in MyoD and myogenin mRNA expression after 12 h of culture compared to cells cultured in differentiation media alone. MyoD and myogenin immunoreactive proteins in cells cultured in differentiation medium supplemented with insulin were quite low compared to those in control culture. Supplementation of the differentiation media containing insulin with LY294002 (a PI3K inhibitor) induced myoblast differentiation. A significant increase in MyoD and myogenin mRNA expression was observed in these cells after incubation for 12 h, and the level of expression was similar to that of control cells incubated with differentiation media alone. The DNA content and the phosphor-Erk1/2 protein level were increased by the addition of insulin to the differentiation medium. These results suggest that insulin and its signaling pathway play an inhibitory role in chick myoblast differentiation. A high level of Pax7 mRNA was observed in the skeletal muscle of 3-day-old chicks administered insulin or tolbutamide at 1-day-of-age. In addition, body weight at 21 and 50 days-of-age was significantly greater for chickens administered insulin or tolbutamide at 1-day-of-age than for control chickens. These results detail not only species-specific differences in insulin action for myoblasts but also provide novel information that may be used for the improvement of chicken meat production.

Selenium Regulates Gene Expression of Selenoprotein W in Chicken Skeletal Muscle System

Selenoprotein W (SelW) is abundantly expressed in skeletal muscles of mammals and necessary for the metabolism of skeletal muscles. However, its expression pattern in skeletal muscle system of birds is still uncovered. Herein, to investigate the distribution of SelW mRNA in chicken skeletal muscle system and its response to different selenium (Se) status, 1-day-old chickens were exposed to various concentrations of Se as sodium selenite in the feed for 35 days. In addition, myoblasts were treated with different concentrations of Se in the medium for 72 h. Then the levels of SelW mRNA in skeletal muscles (wing muscle, pectoral muscle, thigh muscle) and myoblasts were determined on days 1, 15, 25, and 35 and at 0, 24, 48, and 72 h, respectively. The results showed that SelW was detected in all these muscle components and it increased both along with the growth of organism and the differentiation process of myoblasts. The thigh muscle is more responsive to Se intake than the other two skeletal muscle tissues while the optimal Se supplementation for SelW mRNA expression in chicken myoblasts was 10(-7) M. In summary, Se plays important roles in the development of chicken skeletal muscles. To effect optimal SelW gene expression, Se must be provided in the diet and the media in adequate amounts and neither at excessive nor deficient levels.

Prdm1 (Blimp-1) and the Expression of Fast and Slow Myosin Heavy Chain Isoforms during Avian Myogenesis In Vitro

BackgroundMultiple types of fast and slow skeletal muscle fibers form during early embryogenesis in vertebrates. In zebrafish, formation of the earliest slow myofibers in fin muscles requires expression of the zinc-finger transcriptional repressor Prdm1 (also known as Blimp1). To further understand how the role of Prdm1 in early myogenesis may vary through evolution and during development, we have now analyzed Prdm1 expression in the diverse types of myotubes that form in culture from somitic, embryonic, and fetal chicken myoblasts.Principal FindingsIn cultures of somitic, embryonic limb, and fetal limb chicken cells, we found that Prdm1 was expressed in all of the differentiated muscle cells that formed, including those that expressed only fast myosin heavy chain isoforms, as well as those that co-expressed both fast and slow myosin heavy chain isoforms. Prdm1 was also expressed in Pax7-positive myoblasts, as well as in non-myogenic cells in the cultures. Furthermore, though all differentiated cells in control somite cultures co-expressed fast and slow myosin heavy chains, antisense knockdown of Prdm1 expression inhibited the formation of these co-expressing cells in somite cultures.ConclusionsIn chicken myogenic cell cultures, Prdm1 was expressed in most Pax7-positive myoblasts and in all differentiated muscle cells, irrespective of the developmental stage of cell donor or the pattern of fast and slow myosin heavy chains expressed in the differentiated cells that were formed. Thus, Prdm1 was expressed in myogenic cells prior to terminal differentiation; and, after differentiation, Prdm1 expression was not limited to cells that expressed slow myosin heavy chain isoforms. In addition, Prdm1 appeared to be required for differentiation of the somitic myocytes, which are the earliest myocytes to form in the avian embryo.

The myostatin‐induced E3 ubiquitin ligase RNF13 negatively regulates the proliferation of chicken myoblasts

The ubiquitin ligase RING finger protein 13 gene (RNF13) was first identified in a screen for genes whose expression is regulated by myostatin in chicken fetal myoblasts. In this study, we demonstrate that the RNF13 gene is broadly expressed in many chicken tissues. The expression of RNF13 gradually decreases during skeletal myogenesis, and myostatin up-regulates RNF13 expression at both the transcriptional and translational levels. Interestingly, ectopic expression of RNF13 inhibits cell proliferation and suppresses the expression of the myogenic genes MyoD and Caveolin-3 in muscle cells. Moreover, recently, we have reported that RNF13 is a RING-type E3 ubiquitin ligase. In this report, we provide experimental evidence to show that mutations disrupting the RING finger abolish the growth-suppressive activity of RNF13, indicating that its E3 ligase activity is required for the inhibition of cell proliferation. Taken together, our findings show that RNF13 functions as an E3 ubiquitin ligase to negatively regulate cell proliferation.