Search

The development of mammalian skeletal muscle is a highly complex process involving multiple molecular interactions. As a prevalent RNA modification, N6-methyladenosine (m6A) regulates the expression of target genes to affect mammalian development. Nevertheless, it remains unclear how m6A participates in the development of goat muscle. In this study, methyltransferase 3 (METTL3) was significantly enriched in goat longissimus dorsi (LD) tissue. In addition, the global m6A modification level and differentiation of skeletal muscle satellite cells (MuSCs) were regulated by METTL3. By performing mRNA-seq analysis, 8050 candidate genes exhibited significant changes in expression level after the knockdown of METTL3 in MuSCs. Additionally, methylated RNA immunoprecipitation sequencing (MeRIP-seq) illustrated that myocyte enhancer factor 2c (MEF2C) mRNA contained m6A modification. Further experiments demonstrated that METTL3 enhanced the differentiation of MuSCs by upregulating m6A levels and expression of MEF2C. Moreover, the m6A reader YTH N6-methyladenosine RNA binding protein C1 (YTHDC1) was bound and stabilized to MEF2C mRNA. The present study reveals that METTL3 enhances myogenic differentiation in MuSCs by regulating MEF2C and provides evidence of a post-transcriptional mechanism in the development of goat skeletal muscle. [ABSTRACT FROM AUTHOR]

Human antigen R (HuR) is an RNA-binding protein that contributes to a wide variety of biological processes and diseases. HuR has been demonstrated to regulate muscle growth and development, but its regulatory mechanisms are not well understood, especially in goats. In this study, we found that HuR was highly expressed in the skeletal muscle of goats, and its expression levels changed during longissimus dorsi muscle development in goats. The effects of HuR on goat skeletal muscle development were explored using skeletal muscle satellite cells (MuSCs) as a model. The overexpression of HuR accelerated the expression of myogenic differentiation 1 (MyoD), Myogenin (MyoG), myosin heavy chain (MyHC), and the formation of myotubes, while the knockdown of HuR showed opposite effects in MuSCs. In addition, the inhibition of HuR expression significantly reduced the mRNA stability of MyoD and MyoG. To determine the downstream genes affected by HuR at the differentiation stage, we conducted RNA-Seq using MuSCs treated with small interfering RNA, targeting HuR. The RNA-Seq screened 31 upregulated and 113 downregulated differentially expressed genes (DEGs) in which 11 DEGs related to muscle differentiation were screened for quantitative real-time PCR (qRT-PCR) detection. Compared to the control group, the expression of three DEGs (Myomaker, CHRNA1, and CAPN6) was significantly reduced in the siRNA-HuR group (p < 0.01). In this mechanism, HuR bound to Myomaker and increased the mRNA stability of Myomaker. It then positively regulated the expression of Myomaker. Moreover, the rescue experiments indicated that the overexpression of HuR may reverse the inhibitory impact of Myomaker on myoblast differentiation. Together, our findings reveal a novel role for HuR in promoting muscle differentiation in goats by increasing the stability of Myomaker mRNA. [ABSTRACT FROM AUTHOR]

The long non-coding RNAs (lncRNAs) are emerging as essential regulators of the growth and development of skeletal muscles. However, little is known about the expression profiles of lncRNAs during the proliferation and differentiation of skeletal muscle satellite cells (MuSCs) in goats. In this study, we investigate potential regulatory lncRNAs that govern muscle development by performing lncRNA expression profiling analysis during the proliferation (cultured in the growth medium, GM) and differentiation (cultured in the differentiation medium, DM1/DM5) of MuSCs. In total, 1001 lncRNAs were identified in MuSC samples, and 314 differentially expressed (DE) (FDR < 0.05, |log2FC| > 1) lncRNAs were screened by pairwise comparisons from three comparison groups (GM-vs-DM1, GM-vs-DM5, DM1-vs-DM5). Moreover, we identified the cis-, trans-, and antisense-regulatory target genes of DE lncRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that these target genes were significantly enriched in muscle development-related GO terms and KEGG pathways. In addition, the network of interactions between DE lncRNAs and their target genes was identified, which included well-known myogenesis regulators such as Myogenic differentiation 1 (MyoD), Myogenin (MyoG), and Myosin heavy chain (MyHC). Meanwhile, competing endogenous RNA (ceRNA) network analysis showed that 237 DE lncRNAs could bind to 329 microRNAs (miRNAs), while miRNAs could target 564 mRNAs. Together, our results provide a genome-wide resource of lncRNAs that may contribute to myogenic differentiation in goats and lay the groundwork for future investigation into their functions during skeletal muscle development. [ABSTRACT FROM AUTHOR]

As a well-known cancer-related miRNA, miR-193b-3p is enriched in skeletal muscle and dysregulated in muscle disease. However, the mechanism underpinning this has not been addressed so far. Here, we probed the impact of miR-193b-3p on myogenesis by mainly using goat tissues and skeletal muscle satellite cells (MuSCs), compared with mouse C2C12 myoblasts. miR-193b-3p is highly expressed in goat skeletal muscles, and ectopic miR-193b-3p promotes MuSCs proliferation and differentiation. Moreover, insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1) is the most activated insulin signaling gene when there is overexpression of miR-193b-3p; the miRNA recognition element (MRE) within the IGF1BP1 3′ untranslated region (UTR) is indispensable for its activation. Consistently, expression patterns and functions of IGF2BP1 were similar to those of miR-193b-3p in tissues and MuSCs. In comparison, ectopic miR-193b-3p failed to induce PAX7 expression and myoblast proliferation when there was IGF2BP1 knockdown. Furthermore, miR-193b-3p destabilized IGF2BP1 mRNA, but unexpectedly promoted levels of IGF2BP1 heteronuclear RNA (hnRNA), dramatically. Moreover, miR-193b-3p could induce its neighboring genes. However, miR-193b-3p inversely regulated IGF2BP1 and myoblast proliferation in the mouse C2C12 myoblast. These data unveil that goat miR-193b-3p promotes myoblast proliferation via activating IGF2BP1 by binding to its 3′ UTR. Our novel findings highlight the positive regulation between miRNA and its target genes in muscle development, which further extends the repertoire of miRNA functions. [ABSTRACT FROM AUTHOR]

The proliferation and differentiation of mammalian skeletal muscle satellite cells (MuSCs) are highly complicated. Apart from the regulatory signaling cascade driven by the protein-coding genes, non-coding RNAs such as microRNAs (miRNA) and circular RNAs (circRNAs) play essential roles in this biological process. However, circRNA functions in MuSCs proliferation and differentiation remain largely to be elucidated. Here, we screened for an exonic circTCF4 based on our previous RNA-Seq data, specifically expressed during the development of the longest dorsal muscle in goats. Subsequently, the circular structure and whole sequence of circTCF4 were verified using Sanger sequencing. Besides, circTCF4 was spatiotemporally expressed in multiple tissues from goats but strikingly enriched in muscles. Furthermore, circTCF4 suppressed MuSCs proliferation and differentiation, independent of AGO2 binding. Finally, we conducted Poly(A) RNA-Seq using cells treated with small interfering RNA targeting circTCF4 and found that circTCF4 would affect multiple signaling pathways, including the insulin signaling pathway and AMPK signaling pathway related to muscle differentiation. Our results provide additional solid evidence for circRNA regulating skeletal muscle formation. [ABSTRACT FROM AUTHOR]

Prostaglandin (PG) signaling regulates a wide variety of physiological and pathological processes, including body temperature, cardiovascular homeostasis, reproduction, and inflammation. Recent studies have revealed that PGs play pivotal roles in embryo development, ciliogenesis, and organ formation. Prostaglandin E2 (PGE2) and its receptor EP4 modulate ciliogenesis by increasing the anterograde intraflagellar transport. Many G‐protein‐coupled receptors (GPCRs) including EP4 are localized in cilia for modulating cAMP signaling under various conditions. During development, PGE2 signaling regulates embryogenesis, hepatocyte differentiation, hematopoiesis, and kidney formation. Prostaglandins are also essential for skeletal muscle repair. This review outlines recent advances in understanding the functions and mechanisms of prostaglandin signaling in ciliogenesis, embryo development, and organ formation. [ABSTRACT FROM AUTHOR]

Cell Res. 2002; 12: 9-18. 10.1038/sj.cr.7290105 41 Xie S.J., Li J.H., Chen H.F., Tan Y.Y., Liu S.R., Zhang Y., Xu H., Yang J.H., Liu S., Zheng L.L. Inhibition of the JNK/MAPK signaling pathway by myogenesis-associated miRNAs is required for skeletal muscle development. Many gene expression profiling studies in agriculturally significant animals have been carried out since the introduction of high-throughput sequencing tools, and many genes or factors associated to skeletal muscle development have been found [[8], [10]]. Although progress has been made in understanding how genes work during myogenesis in pigs [[8], [10]], cattle [[27]], goats [[11], [28]], and sheep [[9]], our understanding of the genes involved in goat skeletal muscle development is still limited and incomplete. Among them, we indeed identified many terms related to muscle development, such as muscle structure development, muscle tissue development, muscle contraction, muscle cell development, and muscle cell differentiation. [Extracted from the article]

Myogenesis is a complex process controlled by several coding and non-coding RNAs (ncRNAs), such as circular RNAs (circRNAs) that are known to function as endogenous microRNAs (miRNAs) sponges. Cerebellar Degeneration-Related protein 1 antisense (CDR1as) is the most spotlighted circRNA that is known as an miR-7 sponge, which has bloomed circRNAs' research in animal disease and physiology. Here, we screened for miRNAs and mRNA associated with CDR1as and further characterized their regulatory function during muscle differentiation. We found that a total of 43 miRNAs (including miR-107-3p, miR-125b-5p, miR-140-5p, miR-29a-3p, and miR-27a-3p upregulated) and 789 mRNAs (including ANGPT1, E2F2, CCN1, FGFR1, and MEF2C downregulated) were differentially expressed in goat skeletal muscle satellite cells (SMSCs). Further, knockdown of CDR1as and ANGPT1 inhibited SMSCs differentiation. miR-27a-3p was differentially upregulated after the knockdown of CDR1as in SMSCs. Overexpressed miR-27a-3p decreased SMSCs differentiation. Via RNAhybrid and luciferase, miR-27a-3p was identified to regulate ANGPT1. We discovered that miR-27a-3p has an inverse relationship with CDR1as and decreases the expression level of ANGPT1 during SMSCs differentiation. In summary, our study demonstrates that siCDR1as inhibits myoblast differentiation by downregulating ANGPT1 mRNA via miR-27a-3p in SMSCs. [ABSTRACT FROM AUTHOR]

Long noncoding RNAs (lncRNAs) have emerged as essential regulators of skeletal myogenesis, but few myogenesis-associated lncRNAs have been identified and our understanding of their regulatory mechanisms remains limited, particularly in goat. Here, we identified a novel lncRNA, TCONS_00006810 (named lncR-125b), from our previous lncRNA sequencing data on fetal (45, 60, and 105 days of gestation, three biological replicates for each point) and postnatal (3 days after birth, n = 3) goat skeletal muscle, and found that it is highly expressed in skeletal muscle and gradually upregulated during skeletal muscle satellite cell (SMSC) differentiation in goat. Notably, overexpression of lncR-125b accelerated the expression of myogenic differentiation 1 (MyoD 1) and myogenin (MyoG), and the formation of myotubes, and knockdown of lncR-125b showed opposite effects in SMSCs. Results of dual-luciferase assay and quantitative real-time polymerase chain reaction revealed that lncR-125b acts as a molecular sponge for miR-125b and that insulin-like growth factor 2 (IGF2), a critical regulator of skeletal myogenesis, is a direct target gene of miR-125b. Further analyses showed that lncR-125b negatively regulates miR-125b expression and positively regulates IGF2 expression in SMSCs. Mechanistically, lncR-125b promotes SMSC differentiation by functioning as a competing endogenous RNA (ceRNA) for miR-125b to control IGF2 expression. These findings identify lncR-125b as a novel noncoding regulator of muscle cell differentiation and skeletal muscle development in goat. [ABSTRACT FROM AUTHOR]

Glycogen synthase kinase beta (GSK3β) plays an important role in skeletal muscle growth, regeneration, and repair. However, the mechanism of GSK3β regulating MyHC2a expression is currently not clear. In this study, GSK3β inhibition promoted skeletal muscle satellite cells (SMSCs) differentiation and increased expression of MyoD, MyoG, MyHC1, and MyHC2a genes. Then we cloned approximately 1.1 kb of goat MyHC2a gene promoter. The deletion fragment (−514/+55) of MyHC2a promoter exhibited the highest level of promoter activity, and a NFATc2 element in this region was responsible for MyHC2a promoter activity. Treatment of SB216713 significantly decreased the transcriptional activity of the fragment (−514/+55). Furthermore, GSK3β inhibition had no effect on the luciferase activity of MyHC2a promoter after mutating the NFATc2‐binding site. These results demonstrated that GSK3β inhibition promoted SMSCs differentiation and regulated the MyHC2a gene expression through NFATc2 in goat‐differentiated SMSCs. [ABSTRACT FROM AUTHOR]

The important roles of myogenic regulatory factors ( MRFs) have been well addressed in the process of mammalian skeletal myogenesis, while limited research has been performed in small ruminants. Furthermore, the effects of gender on the development of skeletal muscle and MRFs expression remain unknown. In this study, we identified the caprine Myf5, Myf6, MyoD and myogenin genes and quantified their expressions at six different postnatal time points by real-time RT-PCR. The sex has a marked effect on the expression differences of Myf5, MyoD and myogenin in the five investigated skeletal muscles, while minor influence on the expression difference of Myf6 except for Semitendinosus and Quadriceps femoris tissues ( P < 0.001). The histological sections of muscles revealed a constant increase of muscle fiber diameter with aging but non-significant differences between genders. We provide novel evidence for MRFs expression in age- and gender-dependent manners, which will contribute to prioritizing these genes as potential candidate genes for trait-associated study and provide a foundation for understanding the molecular control of skeletal muscle growth in goat species. [ABSTRACT FROM AUTHOR]

Highlights • The Nanjiang Brown(NJ) goats had more myofiber cells compared with the Boer goats. • The Boer goats had larger cross-section area compared with the NJ goats. • The SMSCs proliferation potential was higher in NJ goats than in Boer goats. • The SMSCs fusion rate was higher NJ goats than in Boer goats. • The expressions of Pax7, MyoD, MKK3/6, p38MAPK, MEF2C were higher in NJ goat SMSCs. Abstract Here, for the first time, we analyzed skeletal muscle morphology in Boer and Nanjiang Brown (NJ) goats and then evaluated the differences in skeletal muscle satellite cells (SMSCs) proliferation and differentiation ability between the two breeds. Our results revealed that NJ goats had significantly more myofiber cells and a significantly smaller cross-section area compared with the Boer goats (P < 0.01). The Pax7 , p38MAPK and MEF2A were more enriched in all three muscles of NJ goats, while the expressions of MyoG, TAK1 , and MKK6 presented the opposite tendency. Additionally, MEF2C, MyoD and MKK3 exhibited somewhat muscle-type specific expression patterns. During proliferation there were significantly more SMSCs in NJ goats compared with Boer goats on days 3, 4, and 5 (P < 0.05). Moreover, the SMSCs fusion rate was higher in NJ goats than in Boer goats during the entire differentiation stage, though only significant on the 6th day (P < 0.05). The Pax7 , MyoD , MKK3/6 , p38MAPK , and MEF2C expression levels in NJ goat SMSCs were significantly higher than those in Boer goat SMSCs (P < 0.05). In conclusion, these findings indicate that SMSCs proliferation and differentiation potential were higher in NJ goats than in Boer goats, which possibly contribute to the meat-produce difference between the two breeds. [ABSTRACT FROM AUTHOR]

• Cold exposure increased energy metabolism, resulting in decreased glycogen content in skeletal muscle of newborn goats. • Transcriptome analysis found that cold exposure up-regulated the expression levels of genes related to glucose uptake, glycolysis, and fatty acid and TG synthesis, promoting lipid deposition in skeletal muscle. • Cold exposure induced mitophagy in skeletal muscle. Cold exposure is a common stressor for newborn goats. Skeletal muscle plays an important role in maintaining whole-body homeostasis of glucose and lipid metabolism. However, the molecular mechanisms underlying regulation of skeletal muscle of newborn goats by cold exposure remains unclear. In this study, we found a significant increase (P < 0.01) in serum glucagon levels after 24 h of cold exposure (COLD, 6°C), while glucose and insulin concentrations were significantly decreased (P < 0.01) compared to room temperature (RT, 25°C). Additionally, we found that cold exposure reduced glycogen content (P < 0.01) in skeletal muscle. Pathway enrichment analysis revealed that cold exposure activated skeletal muscle glucose metabolism pathways (including insulin resistance and the insulin signaling pathway) and mitophagy-related pathways. Cold exposure up-regulated the expression of genes involved in fatty acid and triglyceride synthesis, promoting skeletal muscle lipid deposition. Notably, cold exposure induced mitophagy in skeletal muscle. [ABSTRACT FROM AUTHOR]

The elaborate interplay of coding and noncoding factors governs muscle growth and development. Here, we reported a mutual activation between long noncoding RNA (lncRNA) H19 and MyoD (myogenic determination gene number 1) in the muscle process. We successfully cloned the two isoforms of goat H19, which were significantly enriched and positively correlated with MyoD transcripts in skeletal muscles or differentiating muscle satellite cells (MuSCs). To systematically screen genes altered by H19, we performed RNA-seq using cDNA libraries of differentiating H19-deficiency MuSCs and consequently anchored MyoD as the critical genes in mediating H19 function. Intriguingly, some transcripts of MyoD and H19 overlapped in the cytoplasm, which was dramatically damaged when the core complementary nucleotides were mutated. Meanwhile, MyoD RNA successfully pulled down H19 in MS2-RIP experiments. Furthermore, HuR could bind both H19 and MyoD transcripts, while H19 or its truncated mutants successfully stabilized MyoD mRNA, with or without HuR deficiency. In turn, novel functional MyoD protein-binding sites were identified in the promoter and exons of the H19 gene. Our results suggest that MyoD activates H19 transcriptionally, and RNA-RNA hybridization is critical for H19-promoted MyoD expression, which extends our knowledge of the hierarchy of regulatory networks in muscle growth. • H19 promotes differentiation of goat skeletal muscle satellite cells (MuSCs). • H19 partially colocalizes with MyoD transcripts. • H19 significantly stabilizes MyoD mRNA. • MyoD activates H19 by binding on its promoter and exons. [ABSTRACT FROM AUTHOR]

Though muscle characteristics in the second half of gestation closely relate to quantity and quality of postnatal muscle, far fewer studies have examined breed differences in prenatal sheep muscle. This study seeks to better understand anatomic prenatal muscle differences and its underpinning mechanisms between two contrasting sheep breeds. A total of 38 sheep fetuses, including 20 Texel with pronounced muscularity and 18 Ujumqin, an indigenous fat-tailed sheep characterized by their hardiness, fetuses aged at up to five time-points from embryonic day (E) 70-135 of gestation were sacrificed and their organs and muscles dissected at 12 anatomical locations from the right side of each animal. Whole Texel fetuses weighed more especially at E70 and E85 (both 1.7-fold, P <0.001) with heavier internal organs ( P <0.05), than age-matched Ujumqin fetuses. Texel fetuses also contained more muscle, including aggregate-excised muscles (70% before E100, P <0.001) and muscle proportion (~0.5%, P <0.05), larger (~20%) and more total muscle fibers, greater muscle size (20% loin muscle area, P =0.008), and a lower proportion of type I muscle fibers (Texel, 18.85%; Ujumqin, 25.23%) at most anatomical locations. Notably, in Texel fetuses from E70 to E85, sharply decreased myofiber number and significantly augmented fiber size were accompanied by the molecular events resembled those in myoblast fusing of Ujumqin sheep (E85-E100), which indicate this period (E70-E85) is pivotal for Texel myoblast fusing. Altogether, these results suggest that comparing with obese animal, the muscular superiority in lean breed largely establishes at middle gestation, although they share the similar regulatory gene network. The present study provides a novel insight into mammalian myogenesis. [ABSTRACT FROM AUTHOR]

MicroRNAs are a class of small, non-coding RNAs that participate in the regulation of diverse biological processes including skeletal muscle development. However, the underlying molecular mechanisms are not fully understood, particularly in goat. Here, we identified goat miR-101a as a novel myogenic microRNA mediating myogenic differentiation. The expression of miR-101a was enriched in goat skeletal muscles and up-regulated during satellite cell differentiation. After transfection with a miR-101a mimic and culturing in differentiation medium, satellite cell differentiation was promoted, accompanied by a significant increase in mRNA expression of the myogenic marker, MyoG , and decreased expression of MyoD . In contrast, blocking the function of miR-101a with a 2′-O-methylated antisense oligonucleotide inhibitor repressed satellite cell differentiation. However, both gain- and loss-of-function studies demonstrated that miR-101a had no significant influence on satellite cell proliferation. Therefore, our results provide a new insight on miR-101a in the regulation of goat skeletal muscle satellite cell proliferation and differentiation. [ABSTRACT FROM AUTHOR]

Abstract: Actin capping protein beta (CAPZB) protein was identified with considerable differences in the longissimus dorsi muscle between Large White and Meishan pigs using proteomics approach. However, in pigs, the information on CAPZB is very limited. In this study, we cloned and characterized the porcine actin capping protein beta (CAPZB) gene. In addition, we present two novel porcine CAPZB splice variants CAPZB1 and CAPZB2. CAPZB1 was expressed in all twenty tissues. However, CAPZB2 was predominantly expressed in the skeletal muscle and heart. In addition, the two isoforms had different expression profiles during the skeletal muscle development and between breeds. Moreover, the SNP T394G was identified in the coding region of the CAPZB gene, which was significantly associated with the carcass traits including the LFW, CFW, SFT and LEA. Data presented in our study suggests that the CAPZB gene may be a candidate gene of meat production trait and provides useful information for further studies on its roles in porcine skeletal muscle. [Copyright &y& Elsevier]

Abstract: The actinin-associated LIM protein (ALP) subfamily has important functions in cell signal transduction, cell proliferation, and integration of cytoskeletal architecture. To detect their functions in pig skeletal muscle, we cloned and characterized the pig ALP subfamily genes, drew their genomic structure maps, and detected their tissue expression patterns. We identified a new spliced variant of PDLIM3 in pig skeletal muscle and named it as PDLIM3-4, which was only expressed in the heart and skeletal muscle. Our results showed that PDLIM3-4 was expressed in adult pig skeletal muscle with the highest expression level, and both PDLIM3-4 isoform and PDLIM4 had different expression profiles during the prenatal and postnatal stages of skeletal muscle development among the three pig breeds. These studies provide useful information for further research on the functions of pig ALP subfamily genes in skeletal muscle development. [Copyright &y& Elsevier]

Abstract: Intramuscular fat (IMF) shortage causes the lack of juiciness and tenderness of goat meat, while peroxisome proliferator-activated receptor gamma 1 (PPARγ1) and gamma 2 (PPARγ2) play key roles in lipid metabolism. Nevertheless, their expression patterns and the relationship with IMF have been poorly exposed. Using quantitative polymerase chain reaction (qPCR), classical Soxhlet extraction, and in situ hybridization, we demonstrated that among 13 goat tissues, expression of PPARγ1 was dramatically higher than that of PPARγ2 except for lung. We further demonstrated the expression patterns of PPARγ1 and PPARγ2 and their negative association with intramuscular fat content in three goat muscles with kids growing. Meanwhile, PPARγ expression was located in the connective tissues. These results suggest that PPARγ1 is rather active for most tissues of goat, and closely related with the muscular fat metabolism during early postnatal life, but a more direct proof remains to be provided. [Copyright &y& Elsevier]