Developmental overlap between skeletal muscle maturation and perirenal fat brown-to-white transition in goats: Exploring the role of Myf-5.

In mammals, skeletal muscles (SkMs) and adipose tissues regulate energy homeostasis and share developmental origins. Notably, the perirenal adipose tissue (PRAT) depot has been reported to display adipocyte heterogeneity: while some originated from Myogenic factor 5 (Myf-5) expressing progenitors, others did not. Our study examines the expression and distribution of Myf-5 using immunohistochemical staining and western blotting of PRAT, gastrocnemius, and trapezius from goat at various developmental stages. Contrary to earlier beliefs, functionally divergent SkM gastrocnemius and trapezius showed similar Myf-5 expressional pattern. SkM abundantly expresses Myf-5 in developing myocytes which gradually becomes limited to the nucleus of myogenic stem cells and is retained only in a few differentiated postnatal fibers. During the same period, PRAT displays a unique brown-to-white transition. PRAT exhibited an elevated expression of Myf-5 during prenatal periods, which declines thereafter and becomes negligible during adulthood where it gets fully enriched white adipocytes. The reduction of Myf-5 during the neonatal period was common to all three tissues. However, Myf-5 expression was retained in some of the differentiated myofibers while it was undetectable in adult PRAT. These observations suggest a possible developmental interplay between adipose tissue and SkM where Myf-5 might be a major regulator.

A dual-color PAX7 and MYF5 in vivo reporter to investigate muscle stem cell heterogeneity in regeneration and aging.

Increasing evidence suggests that the muscle stem cell (MuSC) pool is heterogeneous. In particular, a rare subset of PAX7-positive MuSCs that has never expressed the myogenic regulatory factor MYF5 displays unique self-renewal and engraftment characteristics. However, the scarcity and limited availability of protein markers make the characterization of these cells challenging. Here, we describe the generation of StemRep reporter mice enabling the monitoring of PAX7 and MYF5 proteins based on equimolar levels of dual nuclear fluorescence. High levels of PAX7 protein and low levels of MYF5 delineate a deeply quiescent MuSC subpopulation with an increased capacity for asymmetric division and distinct dynamics of activation, proliferation, and commitment. Aging primarily reduces the MYF5Low MuSCs and skews the stem cell pool toward MYF5High cells with lower quiescence and self-renewal potential. Altogether, we establish the StemRep model as a versatile tool to study MuSC heterogeneity and broaden our understanding of mechanisms regulating MuSC quiescence and self-renewal in homeostatic, regenerating, and aged muscles.

Identification of a Novel Frameshift Variant in MYF5 Leading to External Ophthalmoplegia with Rib and Vertebral Anomalies.

Myogenic transcription factors with a basic helix-loop-helix (bHLH) such as MYOD, myogenin, MRF4, and MYF5 contribute to muscle differentiation and regulation. The MYF5 gene located on chromosome 12 encodes for myogenic factor 5 (MYF5), which has a role in skeletal and extraocular muscle development and rib formation. Variants in MYF5 were found to cause external ophthalmoplegia with rib and vertebral anomalies (EORVA), a rare recessive condition. To date, three homozygous variants in MYF5 have been reported to cause EORVA in six members of four unrelated families. Here, we present a novel homozygous MYF5 frameshift variant, c.596dupA p. (Asn199Lysfs*49), causing premature protein termination and presenting with external ophthalmoplegia, ptosis, and scoliosis in three siblings from a consanguineous family of Pakistani origin. With four MYF5 variants now discovered, genetic testing and paediatric assessment for extra-ocular features should be considered in all cases of congenital ophthalmoplegia.

Genetic Characterization of Myf5 and POU1F1 Genes in Different Egyptian Local Rabbit Breeds and Their Association with Growth Traits.

Genetic characterization and its association with quantitative traits in local breeds are important tools for the genetic improvement and sustainable management of animal genetic resources. Myogenic regulatory factor 5 (MYf5) and POU class 1 homeobox 1 (POU1F1) are candidate genes which play important roles in growth and development of mammals. The present study aims to detect the genetic diversity of the MYf5 and POU1F1 genes in four local Egyptian rabbit breeds and their association with growth traits, using PCR-restriction enzyme (PCR-RFLP), PCR-single-strand conformational polymorphism (PCR-SSCP), and direct sequencing techniques. The results showed that MYF5 exon 1 was observed with two genotypes in Baladi Black (BB), Gabali (GB) and New Zealand White (NZW) breeds while APRI-line (APRI) presented one genotype. The genetic diversity of Myf5 exon 2 between breeds showed two genotypes in APRI compared to three in NZW and four genotypes in BB and GB breeds. The genetic diversity of the POU1F1 gene (intron 5 and partial cds) in different rabbit breeds was two genotypes in NZW and three genotypes in BB, GB, and APRI breeds with different frequencies for each genotype. Based on the statistically significant difference between genes genotypes and growth weight, the results suggested that the genotypes of Myf5 exon 2 (1 and 2) of the BB breed, Myf5 exon 2 genotype 2 of the APRI breed, and genotype 1 of Myf5 exon 1 and genotype 1 of POU1F1 of the NZW breed compared to genotypes for each gene can be considered candidate molecular markers associated with the improvement of growth traits in these breeds.

Differentiation of Human Fetal Muscle Stem Cells from Induced Pluripotent Stem Cells.

Most muscular dystrophies are the result of genetic disorders. There is currently no effective treatment for these progressive diseases except palliative therapy. Muscle stem cells with potent self-renewal and regenerative potential are considered a target for treating muscular dystrophy. Human induced pluripotent stem cells have been expected as a source of MuSCs because of their infinite proliferation potential and less immunogenicity. However, the generation of engraftable MuSCs from hiPSCs is relatively difficult and encounters low efficiency and reproducibility. Here, we introduce a transgene-free protocol of hiPSCs differentiating into fetal MuSCs by identifying them as MYF5-positive cells. Flow cytometry analysis detected around 10% of MYF5-positive cells after 12 weeks of differentiation. Approximately 50 ~ 60% of MYF5-positive cells were positively identified using Pax7 immunostaining. This differentiation protocol is expected to be useful for not only the establishment of cell therapy but also the future drug discovery using patient-derived hiPSCs.

Blastocyst complementation and interspecies chimeras in gene edited pigs.

The only curative therapy for many endstage diseases is allograft organ transplantation. Due to the limited supply of donor organs, relatively few patients are recipients of a transplanted organ. Therefore, new strategies are warranted to address this unmet need. Using gene editing technologies, somatic cell nuclear transfer and human induced pluripotent stem cell technologies, interspecies chimeric organs have been pursued with promising results. In this review, we highlight the overall technical strategy, the successful early results and the hurdles that need to be addressed in order for these approaches to produce a successful organ that could be transplanted in patients with endstage diseases.

Myogenic regulatory factors Myod and Myf5 are required for dorsal aorta formation and angiogenic sprouting.

The vertebrate embryonic midline vasculature forms in close proximity to the developing skeletal muscle, which originates in the somites. Angioblasts migrate from bilateral positions along the ventral edge of the somites until they meet at the midline, where they sort and differentiate into the dorsal aorta and the cardinal vein. This migration occurs at the same time that myoblasts in the somites are beginning to differentiate into skeletal muscle, a process which requires the activity of the basic helix loop helix (bHLH) transcription factors Myod and Myf5. Here we examined vasculature formation in myod and myf5 mutant zebrafish. In the absence of skeletal myogenesis, angioblasts migrate normally to the midline but form only the cardinal vein and not the dorsal aorta. The phenotype is due to the failure to activate vascular endothelial growth factor ligand vegfaa expression in the somites, which in turn is required in the adjacent angioblasts for dorsal aorta specification. Myod and Myf5 cooperate with Hedgehog signaling to activate and later maintain vegfaa expression in the medial somites, which is required for angiogenic sprouting from the dorsal aorta. Our work reveals that the early embryonic skeletal musculature in teleosts evolved to organize the midline vasculature during development.

Mechanisms and strategies to promote cardiac xenotransplantation.

End stage heart failure is a terminal disease, and the only curative therapy is orthotopic heart transplantation. Due to limited organ availability, alternative strategies have received intense interest for treatment of patients with advanced heart failure. Recent studies using gene-edited porcine organs suggest that cardiac xenotransplantation may provide a future source of organs. In this review, we highlight the historical milestones for cardiac xenotransplantation and the gene editing strategies designed to overcome immunological barriers, which have culminated in a recent cardiac pig-to-human xenotransplant. We also discuss recent results of studies on the engineering of human-porcine chimeric organs that may provide an alternative and complementary strategy to overcome some of the major immunological barriers to producing a new source of transplantable organs.

What snakes and caecilians have in common? Molecular interaction units and the independent origins of similar morphotypes in Tetrapoda.

Developmental pathways encompass transcription factors and cis-regulatory elements that interact as transcription factor-regulatory element (TF-RE) units. Independent origins of similar phenotypes likely involve changes in different parts of these units, a hypothesis promisingly tested addressing the evolution of the rib-associated lumbar (RAL) morphotype that characterizes emblematic animals such as snakes and elephants. Previous investigation in these lineages identified a polymorphism in the Homology region 1 [H1] enhancer of the Myogenic factor-5 [Myf5], which interacts with HOX10 proteins to modulate rib development. Here we address the evolution of TF-RE units focusing on independent origins of RAL morphotypes. We compiled an extensive database for H1-Myf5 and HOX10 sequences with two goals: (i) evaluate if the enhancer polymorphism is present in amphibians exhibiting the RAL morphotype and (ii) test a hypothesis of enhanced evolutionary flexibility mediated by TF-RE units, according to which independent origins of the RAL morphotype might involve changes in either component of the interaction unit. We identified the H1-Myf5 polymorphism in lineages that diverged around 340 Ma, including Lissamphibia. Independent origins of the RAL morphotype in Tetrapoda involved sequence variation in either component of the TF-RE unit, confirming that different changes may similarly affect the phenotypic outcome of a given developmental pathway.

dmrt2 and myf5 Link Early Somitogenesis to Left-Right Axis Determination in Xenopus laevis.

The vertebrate left-right axis is specified during neurulation by events occurring in a transient ciliated epithelium termed left-right organizer (LRO), which is made up of two distinct cell types. In the axial midline, central LRO (cLRO) cells project motile monocilia and generate a leftward fluid flow, which represents the mechanism of symmetry breakage. This directional fluid flow is perceived by laterally positioned sensory LRO (sLRO) cells, which harbor non-motile cilia. In sLRO cells on the left side, flow-induced signaling triggers post-transcriptional repression of the multi-pathway antagonist dand5. Subsequently, the co-expressed Tgf-ß growth factor Nodal1 is released from Dand5-mediated repression to induce left-sided gene expression. Interestingly, Xenopus sLRO cells have somitic fate, suggesting a connection between LR determination and somitogenesis. Here, we show that doublesex and mab3-related transcription factor 2 (Dmrt2), known to be involved in vertebrate somitogenesis, is required for LRO ciliogenesis and sLRO specification. In dmrt2 morphants, misexpression of the myogenic transcription factors tbx6 and myf5 at early gastrula stages preceded the misspecification of sLRO cells at neurula stages. myf5 morphant tadpoles also showed LR defects due to a failure of sLRO development. The gain of myf5 function reintroduced sLRO cells in dmrt2 morphants, demonstrating that paraxial patterning and somitogenesis are functionally linked to LR axis formation in Xenopus.

Characterization of myogenic regulatory factors, myod and myf5 from Megalobrama amblycephala and the effect of lipopolysaccharide on satellite cells in skeletal muscle.

Myod and Myf5 are muscle-specific basic helix-loop-helix (bHLH) transcription factors that play essential roles in regulating skeletal muscle development and growth. In order to investigate potential function of myod and myf5 of Megalobrama amblycephala, an economically important freshwater fish species, in the present study, we characterized the sequences and expression profiles of M. amblycephala myod and myf5. The open reading frame (ORF) sequences of myod and myf5 encoded 275 and 240 amino acids, respectively, possessing analogous structure with the highly conserved domains, bHLH and C-terminal helix III domains. Spatio-temporal expression patterns revealed that myod and myf5 were predominant in skeletal muscle with the highest expression in white muscle, and the highest at 10 days post-hatching (dph) and the segmentation period, respectively. Furthermore, we evaluated the effects of lipopolysaccharide (LPS) on the expression of muscle-related genes in white and red muscle, and proliferation and differentiation of satellite cells. The myod, myf5 and pax-7 expression generally increased and then decreased with increase of LPS concentration and treatment time in red muscle, while these genes showed inconsistent expression patterns in white muscle. In addition, LPS administration caused the frequency increase of satellite cells in red and white muscle especially at 3 and 7 days after LPS-injection.

miR-9-5p promotes myogenic differentiation via the Dlx3/Myf5 axis.

MicroRNAs play an important role in myogenic differentiation, they bind to target genes and regulate muscle formation. We previously found that miR-9-5p, which is related to bone formation, was increased over time during the process of myogenic differentiation. However, the mechanism by which miR-9-5p regulates myogenic differentiation remains largely unknown. In the present study, we first examined myotube formation and miR-9-5p, myogenesis-related genes including Dlx3, Myod1, Mef2c, Desmin, MyoG and Myf5 expression under myogenic induction. Then, we detected the expression of myogenic transcription factors after overexpression or knockdown of miR-9-5p or Dlx3 in the mouse premyoblast cell line C2C12 by qPCR, western blot and myotube formation under myogenic induction. A luciferase assay was performed to confirm the regulatory relationships between not only miR-9-5p and Dlx3 but also Dlx3 and its downstream gene, Myf5, which is an essential transcription factor of myogenic differentiation. The results showed that miR-9-5p promoted myogenic differentiation by increasing myogenic transcription factor expression and promoting myotube formation, but Dlx3 exerted the opposite effect. Moreover, the luciferase assay showed that miR-9-5p bound to the 3'UTR of Dlx3 and downregulated Dlx3 expression. Dlx3 in turn suppressed Myf5 expression by binding to the Myf5 promoter, ultimately inhibiting the process of myogenic differentiation. In conclusion, the miR-9-5p/Dlx3/Myf5 axis is a novel pathway for the regulation of myogenic differentiation, and can be a potential target to treat the diseases related to muscle dysfunction.

Single-Cell RNA Sequencing Reveals Heterogeneity of Myf5-Derived Cells and Altered Myogenic Fate in the Absence of SRSF2.

Splicing factor SRSF2 acts as a critical regulator for cell survival, however, it remains unknown whether SRSF2 is involved in myoblast proliferation and myogenesis. Here, knockdown of SRSF2 in myoblasts causes high rates of apoptosis and defective differentiation. Combined conditional knockout and lineage tracing approaches show that Myf5-cre mice lacking SRSF2 die immediately at birth and exhibit a complete absence of mature myofibers. Mutant Myf5-derived cells (tdtomato-positive cells) are randomly scattered in the myogenic and non-myogenic regions, indicating loss of the community effect required for skeletal muscle differentiation. Single-cell RNA-sequencing reveals high heterogeneity of myf5-derived cells and non-myogenic cells are significantly increased at the expense of skeletal muscle cells in the absence of SRSF2, reflecting altered cell fate. SRSF2 is demonstrated to regulate the entry of Myf5 cells into the myogenic program and ensures their survival by preventing precocious differentiation and apoptosis. In summary, SRSF2 functions as an essential regulator for Myf5-derived cells to respond correctly to positional cues and to adopt their myogenic fate.

Maternal nutrition altered embryonic MYOD1, MYF5, and MYF6 gene expression in genetically fat and lean lines of chickens.

OBJECTIVE: The objectives of this study were to evaluate the effects of daily feed intake during the laying period on embryonic myogenic differentiation 1 (MYOD1), myogenic factor 5 (MYF5), and myogenic factor 6 (MYF6) gene expression in genetically fat and lean lines of chickens. METHODS: An experiment in a 2×2 factorial design was conducted with two dietary intake levels (100% and 75% of nutrition recommendation) and two broiler chicken lines (fat and lean). Two lines of hens (n = 384 for each line) at 23th week of age were randomly divided into 4 treatments with 12 replicates of 16 birds. The experiment started at 27th week of age (5% egg rate) and ended at 54th week of age. Hatched eggs from the medium laying period were collected. Real time polymerase chain reaction analysis was used to analyse the MYOD1, MYF5, and MYF6 mRNA levels of E7, E9, E11, E13, and E15 body tissues and E17, E19, and E21 chest and thigh muscle samples. RESULTS: The results indicated that there were significant effects of line, dietary intake, and interactions between them on MYOD1, MYF5, and MYF6 gene mRNA expression levels in embryonic tissues. Low daily feed intake did not change the expression trend of MYOD1 mRNA in either line, but changed the peak values, especially in lean line. Low daily feed intake altered the trend in MYF5 mRNA expression level in both lines and apparently delayed its onset. There was no apparent effect of low daily feed intake on the trends of MYF6 mRNA expression levels in either line, but it significantly changed the values on many embryonic days. CONCLUSION: Maternal nutrient restriction affects myogenesis and is manifested in the expression of embryonic MYOD1, MYF5, and MYF6 genes. Long term selection for fat deposition in broiler chickens changes the pattern and intensity of myogenesis.

Novel Single Nucleotide Polymorphisms and Haplotype of MYF5 Gene Are Associated with Body Measurements and Ultrasound Traits in Grassland Short-Tailed Sheep.

Myogenic factor 5 plays active roles in the regulation of myogenesis. The aim of this study is to expose the genetic variants of the MYF5 and its association with growth performance and ultrasound traits in grassland short-tailed sheep (GSTS) in China. The combination technique of sequencing and SNaPshot revealed seven SNPs in ovine MYF5 from 533 adult individuals (male 103 and female 430), four of which are novel ones located at g.6838G > A, g.6989 G > T, g.7117 C > A in the promoter region and g.9471 T > G in the second intron, respectively. Genetic diversity indexes showed the seven SNPs in low or intermediate level, but each of them conformed HWE (p > 0.05) in genotypic frequencies. Association analysis indicated that g.6838G > A, g.7117 C > A, g.8371 T > C, g.9471 T > G, and g.10044 C > T had significant effects on growth performance and ultrasound traits. The diplotypes of H1H3 and H2H3 had higher body weight and greater body size, and haplotype H3 had better performance on meat production than the others. In addition, the dual-luciferase reporter assay showed that there are two active regions in the MYF5 promoter located at −1799~−1197 bp and −514~−241 bp, respectively, but g.6838G > A and g.7117 C > A were out of the region, suggesting these two SNPs influence the phenotype by other pathway. The results suggest that the MYF5 gene might be applied as a promising candidate of functional genetic marker in GSTS breeding.

TLE4 regulates muscle stem cell quiescence and skeletal muscle differentiation.

Muscle stem (satellite) cells express Pax7, a key transcription factor essential for satellite cell maintenance and adult muscle regeneration. We identify the corepressor transducin-like enhancer of split-4 (TLE4) as a Pax7 interaction partner expressed in quiescent satellite cells under homeostasis. A subset of satellite cells transiently downregulate TLE4 during early time points following muscle injury. We identify these to be activated satellite cells, and that TLE4 downregulation is required for Myf5 activation and myogenic commitment. Our results indicate that TLE4 represses Pax7-mediated Myf5 transcriptional activation by occupying the -111 kb Myf5 enhancer to maintain quiescence. Loss of TLE4 function causes Myf5 upregulation, an increase in satellite cell numbers and altered differentiation dynamics during regeneration. Thus, we have uncovered a novel mechanism to maintain satellite cell quiescence and regulate muscle differentiation mediated by the corepressor TLE4.

Construction of a Porcine Skeletal Muscle-Specific Promoter by Inducing the Seed Region of miR-208a.

Transgenic promoter systems are of great interest for their potential use in gene therapy or production due to their high activity, long term, and cell specificity. Here, in order to obtain promoters with high activity and expressed specifically in skeletal muscle, the MYOD1, MYF5, and MCK were selected as the candidate genes. The truncated promoters were amplified and their activity was verified through dual-luciferase reporter gene test. We used genetic engineering techniques to improve promoter activity by tandemly linking enhancers and promoters or two promoters. Furthermore, synthetic promoter was the most active when two eMCK enhancers and pMCK promoter were cascaded. To improve the tissue specificity of the promoter, the seed region of translational repressor miR-208a was inserted into the downstream of the promoter (pGL3-2eMCK-pMCK-T208-mCherry-EGFP). The results showed that the expression level of target genes decreased significantly (P < 0.05) in myocardium rather than in skeletal muscle. The results of in vivo transfection indicated that tandem transcriptional regulatory elements can increase promoter activity in mice. This work laid the foundation for future research on genetically modified pigs.

Master regulators of skeletal muscle lineage development and pluripotent stem cells differentiation.

In vertebrates, the skeletal muscles of the body and their associated stem cells originate from muscle progenitor cells, during development. The specification of the muscles of the trunk, head and limbs, relies on the activity of distinct genetic hierarchies. The major regulators of trunk and limb muscle specification are the paired-homeobox transcription factors PAX3 and PAX7. Distinct gene regulatory networks drive the formation of the different muscles of the head. Despite the redeployment of diverse upstream regulators of muscle progenitor differentiation, the commitment towards the myogenic fate requires the expression of the early myogenic regulatory factors MYF5, MRF4, MYOD and the late differentiation marker MYOG. The expression of these genes is activated by muscle progenitors throughout development, in several waves of myogenic differentiation, constituting the embryonic, fetal and postnatal phases of muscle growth. In order to achieve myogenic cell commitment while maintaining an undifferentiated pool of muscle progenitors, several signaling pathways regulate the switch between proliferation and differentiation of myoblasts. The identification of the gene regulatory networks operating during myogenesis is crucial for the development of in vitro protocols to differentiate pluripotent stem cells into myoblasts required for regenerative medicine.

The Satellite Cell at 60: The Foundation Years.

The resident stem cell for skeletal muscle is the satellite cell. On the 50th anniversary of its discovery in 1961, we described the history of skeletal muscle research and the seminal findings made during the first 20 years in the life of the satellite cell (Scharner and Zammit 2011, doi: 10.1186/2044-5040-1-28). These studies established the satellite cell as the source of myoblasts for growth and regeneration of skeletal muscle. Now on the 60th anniversary, we highlight breakthroughs in the second phase of satellite cell research from 1980 to 2000. These include technical innovations such as isolation of primary satellite cells and viable muscle fibres complete with satellite cells in their niche, together with generation of many useful reagents including genetically modified organisms and antibodies still in use today. New methodologies were combined with description of endogenous satellite cells markers, notably Pax7. Discovery of the muscle regulatory factors Myf5, MyoD, myogenin, and MRF4 in the late 1980s revolutionized understanding of the control of both developmental and regerenative myogenesis. Emergence of genetic lineage markers facilitated identification of satellite cells in situ, and also empowered transplantation studies to examine satellite cell function. Finally, satellite cell heterogeneity and the supportive role of non-satellite cell types in muscle regeneration were described. These major advances in methodology and in understanding satellite cell biology provided further foundations for the dramatic escalation of work on muscle stem cells in the 21st century.

Interspecies chimeras as a platform for exogenic organ production and transplantation.

Chronic diseases are associated with considerable morbidity and mortality. Therefore, new therapeutic strategies are warranted. Here, we provide a brief review outlining the rationale and feasibility for the generation of intraspecies and interspecies chimeras, which one day may serve as a platform for organ transplantation. These strategies are further associated with consideration of scientific and ethical issues.