Embryonic Muscle Development Research Articles

Fibin is a crucial mitochondrial regulatory gene in skeletal muscle development.

Fin bud initiation factor homolog (Fibin) is a secreted protein that is relatively conserved among species. It is closely related to fin bud development and can regulate a variety of cellular processes. In our previous high-throughput chromosome conformation capture (Hi-C) study of pig embryonic muscle development, it was found that Fibin has high expression and activity during the development of pig primary muscle fibers. Therefore, we speculated Fibin participated in myogenesis severely. Specific deletion of Fibin in mouse skeletal muscle resulted in abnormal primary muscle fiber development during the embryonic period and a substantial decrease in skeletal muscle mass in adulthood. In vitro, knocking out Fibin in C2C12 cells promoted cell proliferation; however, after myogenic induction, cells lacking Fibin had almost no ability to differentiate into myotubes. During myogenic differentiation, loss of Fibin disrupts the normal function of mitochondria and impairs oxidative phosphorylation, resulting in decrease of NADH and FADH in the electron transport chain. Transmission electron microscopy also showed that mitochondrial morphology of Fibin-deficient C2C12 was impaired. In conclusion, our research has unveiled a novel mechanism of myogenesis regulation in mitochondrial function and potential target Fibin, and improved understanding of a broad range of mitochondrial muscle diseases.

Activin Signaling Pathway Specialization During Embryonic and Skeletal Muscle Development in Rainbow Trout (Oncorhynchus mykiss).

Activin signaling is essential for proper embryonic, skeletal muscle, and reproductive development. Duplication of the pathway in teleost fish has enabled diversification of gene function across the pathway but how gene duplication influences the function of activin signaling in non-mammalian species is poorly understood. Full characterization of activin receptor signaling pathway expression was performed across embryonic development and during early skeletal muscle growth in rainbow trout (RBT, Oncorhynchus mykiss). Rainbow trout are a model salmonid species that have undergone two additional rounds of whole genome duplication. A small number of genes were expressed early in development and most genes increased expression throughout development. There was limited expression of activin Ab in RBT embryos despite these genes exhibiting significantly elevated expression in post-hatch skeletal muscle. CRISPR editing of the activin Aa1 ohnolog and subsequent production of meiotic gynogenetic offspring revealed that biallelic disruption of activin Aa1 did not result in developmental defects, as occurs with knockout of activin A in mammals. The majority of gynogenetic offspring exhibited homozygous activin Aa1 genotypes (wild type, in-frame, or frameshift) derived from the mosaic founder female. The research identifies mechanisms of specialization among the duplicated activin ohnologs across embryonic development and during periods of high muscle growth in larval and juvenile fish. The knowledge gained provides insights into potential viable gene-targeting approaches for engineering the activin receptor signaling pathway and establishes the feasibility of employing meiotic gynogenesis as a tool for producing homozygous F1 genome-edited fish for species with long-generation times, such as salmonids.

Establishment and Characterization of a Chicken Myoblast Cell Line.

Skeletal muscle, which is predominantly constituted by multinucleated muscle fibers, plays a pivotal role in sustaining bodily movements and energy metabolism. Myoblasts, which serve as precursor cells for differentiation and fusion into muscle fibers, are of critical importance in the exploration of the functional genes associated with embryonic muscle development. However, the in vitro proliferation of primary myoblasts is inherently constrained. In this study, we achieved a significant breakthrough by successfully establishing a chicken myoblast cell line through the introduction of the exogenous chicken telomerase reverse transcriptase (chTERT) gene, followed by rigorous G418-mediated pressure screening. This newly developed cell line, which was designated as chTERT-myoblasts, closely resembled primary myoblasts in terms of morphology and exhibited remarkable stability in culture for at least 20 generations of population doublings without undergoing malignant transformation. In addition, we conducted an exhaustive analysis that encompassed cellular proliferation, differentiation, and transfection characteristics. Our findings revealed that the chTERT-myoblasts had the ability to proliferate, differentiate, and transfect after multiple rounds of population doublings. This achievement not only furnished a valuable source of homogeneous avian cell material for investigating embryonic muscle development, but also provided valuable insights and methodologies for establishing primary cell lines.

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.

IGF2 promotes the differentiation of chicken embryonic myoblast by regulating mitochondrial remodeling.

Skeletal muscle is crucial for animal movement and posture maintenance, and it serves as a significant source of meat in the livestock and poultry industry. The number of muscle fibers differentiated from myoblast in the embryonic stage is one of the factors determining the content of skeletal muscle. Insulin-like growth factor 2 (IGF2), a well-known growth-promoting hormone, is crucial for embryonic and skeletal muscle growth and development. However, the specific molecular mechanism underlying its impact on chicken embryonic myoblast differentiation remains unclear. To elucidate the molecular mechanism by which IGF2 regulates chicken myoblast differentiation, we manipulated IGF2 expression in chicken embryonic myoblast. The results demonstrated that IGF2 was upregulated during chicken skeletal muscle development and myoblast differentiation. On the one hand, we found that IGF2 promotes mitochondrial biogenesis through the PGC1/NRF1/TFAM pathway, thereby enhancing mitochondrial membrane potential, oxidative phosphorylation, and ATP synthesis during myoblast differentiation. This process is mediated by the PI3K/AKT pathway. On the other hand, IGF2 regulates BNIP3-mediated mitophagy, clearing dysfunctional mitochondria. Collectively, our findings confirmed that IGF2 cooperatively regulates mitochondrial biogenesis and mitophagy to remodel the mitochondrial network and enhance mitochondrial function, ultimately promoting myoblast differentiation.

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.

Abstract LB403: Suppression of antigen presentation in pediatric rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma that is diagnosed based on the tumor cell's resemblance to embryonic or poorly differentiated skeletal muscle progenitors. Multiple immunotherapy clinical trials have found no evidence of immune reactivity against RMS, which has been attributed to the relatively low tumor mutation burden in these cancers. However, we made an unexpected observation that many patient RMS tumors had a near absence of MHC-I expression, a key requisite for anti-tumor CD8+ T lymphocytes and many immunotherapies. In this study, we explore the etiology of low antigen presentation in RMS and systematically evaluate therapeutic interventions that sensitize RMS to immunotherapy. Methods: Surface MHC-I expression was evaluated on pediatric tumor cell lines (n=65) from five major pediatric tumor types. RNA-seq, whole exome and genome sequencing data were analyzed from RMS tumors (total of n=247) and iPSC models of skeletal muscle development (PMID: 32011235). Epigentic drugs targeting DNA methylation (decitabine), EZH2 (tazemetostat), Class I/IV HDACs (mocetinostat), and LSD1 (GSK-LSD1) were used at sub-cytotoxic doses on RMS cell lines (n=10) and analyzed for surface MHC-I expression with flow cytometry and gene expression. T cell cytotoxicity assays were performed using engineered human T cells expressing an optimized TCR which recognizes an HLA*A2:01 restricted peptide from PRAME. Results: RMS cell line models exhibited a range of MHC-I expression with most having low or undetectable MHC-I expression, similar to that of a known MHC-I negative tumor type MYCN-amplified neuroblastoma. Exome sequencing of RMS tumors showed no evidence of loss-of-function alterations in essential antigen processing and presentation (APP) pathway genes. Gene expression profiling by RNAseq and western blotting, however, revealed an absence of core APP genes in many RMS tumors and cell lines. We uncovered that during a specific window of embryonic myogenesis, APP is suppressed and that RMS tumors mimicking this state of development show the deepest loss of APP. Multiple classes of epigenetic drugs were evaluated to rescue APP in RMS. Treatment of RMS using a DNA demethylating agent or HDAC inhibitor exhibited the strongest MHC-I restoration across RMS models with no changes observed using LSD1 or EZH2 inhibitors. As a proof-of-concept, we demonstrate that targeting the RMS antigen PRAME using TCR therapy is enhanced by the inclusion of epigenetic therapy. Conclusions and Significance: We revealed that epigenetic suppression of APP is a new hallmark of RMS and normal embryonic skeletal muscle development. Pharmacological reversal of APP pathway downregulation could be achieved using drugs targeting DNA methylation or class I/IV HDACs, and enhanced T cell cytotoxicity against RMS when used in combination. Overall, these findings may explain the historical lack of RMS responsiveness to immunotherapy and unlocks new immunotherapy strategies against RMS that rely on targeting MHC-I antigens. Citation Format: David Milewski, Hsien-Chao Chou, Vineela Gangalapudi, Meijie Tian, Yong Kim, Young Song, Jun Wei, Javed Khan. Suppression of antigen presentation in pediatric rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB403.

Systematic Identification of Long Noncoding RNAs during Three Key Organogenesis Stages in Zebrafish.

Thousands of lncRNAs have been found in zebrafish embryogenesis and adult tissues, but their identification and organogenesis-related functions have not yet been elucidated. In this study, high-throughput sequencing was performed at three different organogenesis stages of zebrafish embryos that are important for zebrafish muscle development. The three stages were 10 hpf (hours post fertilization) (T1), 24 hpf (T2), and 36 hpf (T3). LncRNA gas5, associated with muscle development, was screened out as the next research target by high-throughput sequencing and qPCR validation. The spatiotemporal expression of lncRNA gas5 in zebrafish embryonic muscle development was studied through qPCR and in situ hybridization, and functional analysis was conducted using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9, CRISPR/Cas9). The results were as follows: (1) A total of 1486 differentially expressed lncRNAs were identified between T2 and T1, among which 843 lncRNAs were upregulated and 643 were downregulated. The comparison with T3 and T2 resulted in 844 differentially expressed lncRNAs, among which 482 lncRNAs were upregulated and 362 lncRNAs were downregulated. A total of 2137 differentially expressed lncRNAs were found between T3 and T1, among which 1148 lncRNAs were upregulated and 989 lncRNAs were downregulated, including lncRNA gas5, which was selected as the target gene. (2) The results of spatiotemporal expression analysis showed that lncRNA gas5 was expressed in almost all detected embryos of different developmental stages (0, 2, 6, 10, 16, 24, 36, 48, 72, 96 hpf) and detected tissues of adult zebrafish. (3) After lncRNA gas5 knockout using CRISPR/Cas9 technology, the expression levels of detected genes related to muscle development and adjacent to lncRNA gas5 were more highly affected in the knockout group compared with the control group, suggesting that lncRNA gas5 may play a role in embryonic muscle development in zebrafish. (4) The results of the expression of the skeletal myogenesis marker myod showed that the expression of myod in myotomes was abnormal, suggesting that skeletal myogenesis was affected after lncRNA gas5 knockout. The results of this study provide an experimental basis for further studies on the role of lncRNA gas5 in the embryonic skeletal muscle development of zebrafish.

RASGRP1 targeted by H3K27me3 regulates myoblast proliferation and differentiation in mice and pigs.

Skeletal muscle is not only the largest organ in the body that is responsible for locomotion and exercise but also crucial for maintaining the body's energy metabolism and endocrine secretion. The trimethylation of histone H3 lysine 27 (H3K27me3) is one of the most important histone modifications that participates in muscle development regulation by repressing the transcription of genes. Previous studies indicate that the RASGRP1 gene is regulated by H3K27me3 in embryonic muscle development in pigs, but its function and regulatory role in myogenesis are still unclear. In this study, we verify the crucial role of H3K27me3 in RASGRP1 regulation. The gain/loss function of RASGRP1 in myogenesis regulation is performed using mouse myoblast C2C12 cells and primarily isolated porcine skeletal muscle satellite cells (PSCs). The results of qPCR, western blot analysis, EdU staining, CCK-8 assay and immunofluorescence staining show that overexpression of RASGRP1 promotes cell proliferation and differentiation in both skeletal muscle cell models, while knockdown of RASGRP1 leads to the opposite results. These findings indicate that RASGRP1 plays an important regulatory role in myogenesis in both mice and pigs.

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.

The Integration of Genome-Wide DNA Methylation and Transcriptomics Identifies the Potential Genes That Regulate the Development of Skeletal Muscles in Ducks.

DNA methylation is a pivotal epigenetic regulatory mechanism in the development of skeletal muscles. Nonetheless, the regulators responsible for DNA methylation in the development of embryonic duck skeletal muscles remain unknown. In the present study, whole genome bisulfite sequencing (WGBS) and transcriptome sequencing were conducted on the skeletal muscles of embryonic day 21 (E21) and day 28 (E28) ducks. The DNA methylation pattern was found to fall mainly within the cytosine-guanine (CG) context, with high methylation levels in the intron, exon, and promoter regions. Overall, 7902 differentially methylated regions (DMRs) were identified, which corresponded to 3174 differentially methylated genes (DMGs). By using integrative analysis of both WGBS with transcriptomics, we identified 1072 genes that are DMGs that are negatively associated with differentially expressed genes (DEGs). The gene ontology (GO) analysis revealed significant enrichment in phosphorylation, kinase activity, phosphotransferase activity, alcohol-based receptors, and binding to cytoskeletal proteins. The Kyoto Encyclopedia of Genes and Genomes (KEGGs) analysis showed significant enrichment in MAPK signaling, Wnt signaling, apelin signaling, insulin signaling, and FoxO signaling. The screening of enriched genes showed that hyper-methylation inhibited the expression of Idh3a, Got1, Bcl2, Mylk2, Klf2, Erbin, and Klhl38, and hypo-methylation stimulated the expression of Col22a1, Dnmt3b, Fn1, E2f1, Rprm, and Wfikkn1. Further predictions showed that the CpG islands in the promoters of Klhl38, Klf2, Erbin, Mylk2, and Got1 may play a crucial role in regulating the development of skeletal muscles. This study provides new insights into the epigenetic regulation of the development of duck skeletal muscles.

Transcriptomic Analysis on Pectoral Muscle of European Meat Pigeons and Shiqi Pigeons during Embryonic Development.

In avian muscle development, embryonic muscle development determines the number of myofibers after birth. Therefore, in this study, we investigated the phenotypic differences and the molecular mechanism of pectoral muscle development of the European meat pigeon Mimas strain (later called European meat pigeon) and Shiqi pigeon on embryonic day 6 (E6), day 10 (E10), day 14 (E14) and day 1 after birth (P1). The results showed that the myofiber density of the Shiqi pigeon was significantly higher than that of the European meat pigeon on E6, and myofibers with a diameter in the range of 50~100 μm of the Shiqi pigeon on P1 were significantly higher than those of European meat pigeon. A total of 204 differential expressed genes (DEGs) were obtained from RNA-seq analysis in comparison between pigeon breeds at the same stage. DEGs related to muscle development were found to significantly enrich the cellular amino acid catabolism, carboxylic acid catabolism, extracellular matrix receptor interaction, REDOX enzyme activity, calcium signaling pathway, ECM receptor interaction, PPAR signaling pathway and other pathways. Using Cytoscape software to create mutual mapping, we identified 33 candidate genes. RT-qPCR was performed to verify the 8 DEGs selected-DES, MYOD, MYF6, PTGS1, MYF5, MYH1, MSTN and PPARG-and the results were consistent with RNA-seq. This study provides basic data for revealing the distinct embryonic development mechanism of pectoral muscle between European meat pigeons and Shiqi pigeons.

Mustn1 ablation in skeletal muscle results in increased glucose tolerance concomitant with upregulated GLUT expression in male mice.

Glucose homeostasis is closely regulated to maintain energy requirements of vital organs and skeletal muscle plays a crucial role in this process. Mustn1 is expressed during embryonic and postnatal skeletal muscle development and its function has been implicated in myogenic differentiation and myofusion. Whether Mustn1 plays a role in glucose homeostasis in anyway remains largely unknown. As such, we deleted Mustn1 in skeletal muscle using a conditional knockout (KO) mouse approach. KO mice did not reveal any specific gross phenotypic alterations in skeletal muscle. However, intraperitoneal glucose tolerance testing (IPGTT) revealed that 2-month-old male KO mice had significantly lower glycemia than their littermate wild type (WT) controls. These findings coincided with mRNA changes in genes known to be involved in glucose metabolism, tolerance, and insulin sensitivity; 2-month-old male KO mice had significantly higher expression of GLUT1 and GLUT10 transporters, MUP-1 while OSTN expression was lower. These differences in glycemia and gene expression were statistically insignificant after 4 months. Identical experiments in female KO and WT control mice did not indicate any differences at any age. Our results suggest a link between Mustn1 expression and glucose homeostasis during a restricted period of skeletal muscle development/maturation. While this is an observational study, Mustn1's relationship to glucose homeostasis appears to be more complex with a possible connection to other key proteins such as GLUTs, MUP-1, and OSTN. Additionally, our data indicate temporal and sex differences. Lastly, our findings strengthen the notion that Mustn1 plays a role in the metabolic capacity of skeletal muscle.

DNA Demethylation of Myogenic Genes May Contribute to Embryonic Leg Muscle Development Differences between Wuzong and Shitou Geese.

Skeletal muscle development from embryonic stages to hatching is critical for poultry muscle growth, during which DNA methylation plays a vital role. However, it is not yet clear how DNA methylation affects early embryonic muscle development between goose breeds of different body size. In this study, whole genome bisulfite sequencing (WGBS) was conducted on leg muscle tissue from Wuzong (WZE) and Shitou (STE) geese on embryonic day 15 (E15), E23, and post-hatch day 1. It was found that at E23, the embryonic leg muscle development of STE was more intense than that of WZE. A negative correlation was found between gene expression and DNA methylation around transcription start sites (TSSs), while a positive correlation was observed in the gene body near TTSs. It was also possible that earlier demethylation of myogenic genes around TSSs contributes to their earlier expression in WZE. Using pyrosequencing to analyze DNA methylation patterns of promoter regions, we also found that earlier demethylation of the MyoD1 promoter in WZE contributed to its earlier expression. This study reveals that DNA demethylation of myogenic genes may contribute to embryonic leg muscle development differences between Wuzong and Shitou geese.

The Molecular Mechanism of the TEAD1 Gene and miR-410-5p Affect Embryonic Skeletal Muscle Development: A miRNA-Mediated ceRNA Network Analysis.

Muscle development is a complex biological process involving an intricate network of multiple factor interactions. Through the analysis of transcriptome data and molecular biology confirmation, this study aims to reveal the molecular mechanism underlying sheep embryonic skeletal muscle development. The RNA sequencing of embryos was conducted, and microRNA (miRNA)-mediated competitive endogenous RNA (ceRNA) networks were constructed. qRT-PCR, siRNA knockdown, CCK-8 assay, scratch assay, and dual luciferase assay were used to carry out gene function identification. Through the analysis of the ceRNA networks, three miRNAs (miR-493-3p, miR-3959-3p, and miR-410-5p) and three genes (TEAD1, ZBTB34, and POGLUT1) were identified. The qRT-PCR of the DE-miRNAs and genes in the muscle tissues of sheep showed that the expression levels of the TEAD1 gene and miR-410-5p were correlated with the growth rate. The knockdown of the TEAD1 gene by siRNA could significantly inhibit the proliferation of sheep primary embryonic myoblasts, and the expression levels of SLC1A5, FoxO3, MyoD, and Pax7 were significantly downregulated. The targeting relationship between miR-410-5p and the TEAD1 gene was validated by a dual luciferase assay, and miR-410-5p can significantly downregulate the expression of TEAD1 in sheep primary embryonic myoblasts. We proved the regulatory relationship between miR-410-5p and the TEAD1 gene, which was related to the proliferation of sheep embryonic myoblasts. The results provide a reference and molecular basis for understanding the molecular mechanism of embryonic muscle development.

The Essential Function of miR-5739 in Embryonic Muscle Development.

Embryologically, mesodermal development is closely related to the development of various organs such as muscles, blood vessels, and hearts, which are the main organs that make up the body. However, treatment for mesoderm developmental disorders caused by congenital or acquired factors has so far relied on surgery and drug treatment for symptom relief, and more fundamentally, treatment for mesoderm developmental disorders is needed. In our study, microRNA (miRNA), which plays an important role in the mesoderm development process, was identified and the developmental function was evaluated. miRNAs consist of small nucleotides, which act as transcription factors that bind to the 3' untranslated region and suppressed target gene expression. We constructed the human embryonic stem cell (hESC) knockout cell line and analyzed the function and characteristics of miR-5739, which plays an important role in mesoderm lineage. miR-5739 acts as a transcription factor targeting SMA, Brachyury T, Hand1, which controls muscle proliferation and differentiation, and KDR gene, which regulates vessel formation in vitro. In vivo results suggest a role in regulating muscle proliferation and differentiation. Gene ontology analysis confirmed that the miR-5739 is closely related to genes that regulate muscle and vessel proliferation and differentiation. Importantly, abnormal expression of miR-5739 was detected in somatic cells derived from patients with congenital muscle disease. Our study demonstrate that miR-5739 gene function significantly affects transcriptional circuits that regulate muscle and vascular differentiation during embryonic development.

Histopathological and immunohistochemical study to determine the effects of monosodium glutamate on skeletal muscle development in chickens

The aim of this report was to investigate the effects of MSG administration on embryonic skeletal muscle development in chickens, using histological, histopathological, and immunohistochemical methods. A total of 410 fertilized chicken eggs were used and were divided into five groups: negative control, vehicle control, low-dose group (0.12 mg/g egg MSG), medium-dose group (0.6 mg/g egg MSG), and high-dose group (1.2 mg/g egg MSG). At incubation days 15, 18, and 21, eggs from each group were opened and six live embryos were obtained. The embryos were sacrificed by decapitation, and skeletal muscle tissue samples (musculus fibularis longus and musculus sternocoracoideus pectoralis) were obtained. Sections were stained with hematoxylin-eosin. Moreover, caspase-3 reactivity was determined using the immunohistochemical method. As a result, muscle development was delayed in the MSG groups and the number of caspase-3-positive cells was higher (p < 0.05) than in the controls. Histopathological examinations revealed degenerative and necrotic changes in the skeletal muscle. Muscle degeneration (edema, muscle fiber degeneration, Zenker’s necrosis, and mononuclear cell infiltrations) were observed in all groups, except for the control groups. It was concluded that MSG may have adversely affected the development of the skeletal muscle.

Characteristics and functions of DNA N(6)-methyladenine in embryonic chicken muscle development

DNA N(6)-methyladenine (DNA-6mA) is a new epigenetic mark in eukaryotes, the distribution and functions of which in genomic DNA remain unknown. Although recent studies have suggested that 6mA is present in multiple model organisms and is dynamically regulated during development, the genomic features of 6mA in avian species have yet to be elucidated. 6mA immunoprecipitation sequencing approach was used to analysis the distribution and function of 6mA in the muscle genomic DNA during embryonic chicken development. 6mA immunoprecipitation sequencing was combined with transcriptomic sequencing to reveal the role of 6mA in the regulation of gene expression and to explore possible pathways by which 6mA is involved in muscle development. We here provide evidence that 6mA modification exists widely throughout the chicken genome, and show preliminary data regarding genome-wide distribution of this epigenetic mark. Gene expression was shown to be inhibited by 6mA modification in promoter regions. In addition, the promoters of some genes related to development were modified by 6mA, indicating that 6mA may be involved in embryonic chicken development. Furthermore, 6mA may participate in muscle development and immune function by regulating HSPB8 and OASL expression. Our study improves our understanding of the distribution and function of 6mA modification in higher organisms and provide new information about differences between mammals and other vertebrates. These findings demonstrate an epigenetic role for 6mA in gene expression and potential involvement in chicken muscle development. Furthermore, the results suggest a potential epigenetic role for 6mA in avian embryonic development.

Polymorphism, Expression, and Structure Analysis of a Key Gene ARNT in Sheep (Ovis aries).

Growth traits are influential factors that significantly affects the development of the sheep industry. A previous TMT proteomic analysis found that a key protein in the HIF signaling pathway, ARNT, may influence embryonic skeletal muscle growth and development in sheep. The purpose of this study was to better understand the association between the polymorphisms of ARNT and growth traits of sheep, and the potential function of ARNT. Real-time qPCR (qRT-PCR) of ARNT was carried out to compare its expression in different developmental stages of the muscle tissues and primary myoblasts in the Hu, Chinese merino, and Gangba sheep. The genetic variance of ARNT was detected using the Illumina Ovine SNP 50 K and 600 K BeadChip in the Hu and Ujimqin sheep populations, respectively. The CDS sequence of the ARNT gene was cloned in the Hu sheep using PCR technology. Finally, bioinformatic analytical methods were applied to characterize the genes and their hypothetical protein products. The qRT-PCR results showed that the ARNT gene was expressed significantly in the Chinese merino embryo after 85 gestation days (D85) (p < 0.05). Additionally, after the sheep were born, the expression of ARNT was significant at the weaning stage of the Hu sheep (p < 0.01). However, there was no difference in the Gangba sheep.In addition, six SNP loci were screened using 50 K and 600 K BeadChip. We found a significant association between rs413597480 A > G and the Hu sheep weight at weaning and backfat thickness in the 5-month-old sheep (p < 0.05), and four SNP loci (rs162298018 G > C, rs159644025 G > A, rs421351865 G > A, and rs401758103 A > G) were also associated with growth traits in the Ujimqin sheep (p < 0.05). Interestingly, we found that a G > C mutation at 1948 bp in the cloned ARNT CDS sequence of the Hu sheep was the same locus mutation as rs162298018 G > C identified using the 600 K BeadChip, which resulted in a nonconservative missense point mutation, leading to a change from proline to alanine and altering the number of DNA, protein-binding sites, and the α-helix of the ARNT protein. There was a strong linkage disequilibrium between rs162298018 G > C and rs159644025 G > A, and the ARNT protein was conserved among the goat, Hu sheep, and Texel sheep. And, we propose that a putative molecular marker for growth and development in sheep may be the G > C mutation at 1948 bp in the CDS region of the ARNT gene. Our study systematically analyzed the expression, structure, and function of the ARNT gene and its encoded proteins in sheep. This provides a basis for future studies of the regulatory mechanisms of the ARNT gene.

Spatial and temporal requirement of Mlp60A isoforms during muscle development and function in Drosophila melanogaster

Many myofibrillar proteins undergo isoform switching in a spatio-temporal manner during muscle development. The biological significance of the variants of several of these myofibrillar proteins remains elusive. One such myofibrillar protein, the Muscle LIM Protein (MLP), is a vital component of the Z-discs. In this paper, we show that one of the Drosophila MLP encoding genes, Mlp60A, gives rise to two isoforms: a short (279 bp, 10 kDa) and a long (1461 bp, 54 kDa) one. The short isoform is expressed throughout development, but the long isoform is adult-specific, being the dominant of the two isoforms in the indirect flight muscles (IFMs). A concomitant, muscle-specific knockdown of both isoforms leads to partial developmental lethality, with most of the surviving flies being flight defective. A global loss of both isoforms in a Mlp60A-null background also leads to developmental lethality, with muscle defects in the individuals that survive to the third instar larval stage. This lethality could be rescued partially by a muscle-specific overexpression of the short isoform. Genetic perturbation of only the long isoform, through a P-element insertion in the long isoform-specific coding sequence, leads to defective flight, in around 90% of the flies. This phenotype was completely rescued when the P-element insertion was precisely excised from the locus. Hence, our data show that the two Mlp60A isoforms are functionally specialized: the short isoform being essential for normal embryonic muscle development and the long isoform being necessary for normal adult flight muscle function.