Myf5 Gene Expression Research Articles

Early-age heat exposure improved growth performance and heat tolerance in broilers

A total of 440 one-day-old healthy male Arbor Acres broilers were equally assigned to a control group (CTL) and an early-age high-temperature exposure (EHT) group (4 replicates per group, 55 chickens per replicate). At d 3, the broilers in CTL group were reared in the normal temperature 33 ± 1°C, while the broilers in EHT group were exposed to 36 ± 1°C for 24 h. At d 43, all broilers were treated with an acute high temperature 35 ± 1°C for 5 h. The results showed that average daily gain in EHT group was decreased at d 3, but average daily gain in EHT group was increased at d 36 to 42 (P < 0.05). Plasma GLU level in EHT group was lower in broilers at d 7 or facing subsequently high temperature for 5 h (P < 0.05). The relative expression of myogenic differentiation (MyoD) gene in pectoralis major and myogenic factor 5 (Myf5) gene in biceps femoris were significantly improved at d 42 after early-age heat exposure (P < 0.05). Broilers in EHT group have a higher temperature tolerance with a lower mortality than control broilers (P < 0.05). Broilers in EHT group have a lower rectal temperature and a higher comb and ear temperature when facing subsequently acute high temperature than control broilers (P < 0.05). In addition, our study demonstrated that early-age heat exposure significantly decreased the mortality and increased the heat tolerance of broilers when facing an acute short-term heat exposures. Early-age heat exposure increased the process of myogenesis via up-regulating the MyoD and Myf5 gene expression in skeletal muscle, which accelerated average daily gain.

Prehatch thermal manipulation of embryos and posthatch baicalein supplementation increased liver metabolism, and muscle proliferation in broiler chickens

The exposure of broiler chickens to high ambient temperatures causes heat stress (HS), negatively affecting their health and production performance. To mitigate heat stress in broilers, various strategies, including dietary, managerial, and genetic interventions, have been extensively tested with varying degrees of efficacy. For sustainable broiler production, it is imperative to develop an innovative approach that effectively mitigates the adverse effects of HS. Our previous studies have provided valuable insights into the effects of prehatch embryonic thermal manipulation (TM) and posthatch baicalein supplementation on embryonic thermotolerance, metabolism, and posthatch growth performance. This follow-up study investigated the effect of these interventions on gluconeogenesis and lipid metabolism in the liver, as well as muscle proliferation and regeneration capacity in heat-stressed broiler chickens. A total of six-hundred fertile Cobb 500 eggs were incubated for 21 d. After candling, 238 eggs were subjected to TM at 38.5°C with 55% relative humidity (RH) from embryonic day (ED) 12 to 18. These eggs were transferred to the hatcher and kept at a standard temperature (37.5°C) from ED 19 to 21, while 236 eggs were incubated at a controlled temperature (37.5°C) till hatch. After hatching, 180 day-old chicks from both groups were raised in 36 pens treatment (n = 10 birds/pen, 6 replicates per treatment). The treatments were: 1) Control, 2) TM, 3) Control heat stress (CHS), 4) Thermal manipulation heat stress (TMHS), 5) Control heat stress supplement (CHSS), and 6) Thermal manipulation heat stress supplement (TMHSS). Baicalein was added to the treatment group diets starting from d 1. All birds were raised under the standard environment for 21 d, followed by chronic heat stress from d 22 to 35 (32-33 ⁰C for 8 h) in the CHS, TMHS, CHSS, and TMHSS groups. A thermoneutral (22-24⁰C) environment was maintained in the Control and TM groups. RH was constant (50 ± 5%) throughout the trial. In the liver, TM significantly increased (P < 0.05) IGF2 expression. Baicalein supplementation significantly increased (P < 0.05) HSF3, HSP70, SOD1, SOD2, TXN, PRARα, and GHR expression. Moreover, the combination of TM and baicalein supplementation significantly increased (P < 0.05) the expression of HSPH1, HSPB1, HSP90, LPL, and GHR. In the muscle, TM significantly increased (P < 0.05) HSF3 and Myf5 gene expression. TM and baicalein supplementation significantly increased (P < 0.05) the expression of MyoG and significantly (P < 0.05) decreased mTOR and PAX7. In conclusion, the prehatch TM of embryos and posthatch baicalein supplementation mitigated the deleterious effects of HS on broiler chickens by upregulating genes related to liver gluconeogenesis, lipid metabolism, and muscle proliferation.

A Rapid Integrated Detection Platform for Genes Related to Duck Muscle Tissue Development

In duck breeding, the growth and development of skeletal muscle is an important factor influencing the meat production performance of ducks. Therefore, the determination of Myod and Myf5 gene expression in poultry skeletal muscle tissues can help to understand the muscle development of poultry, improve the production performance and feed conversion rate of animal organism, enhance the rapid protein deposition in animal organism, and obtain high quality and quantity of animal livestock products. In this study, a fluorescent PCR assay system for Myod and Myf5 genes was developed, and a dual integrated rapid detection platform suitable for detecting Myod and Myf5 genes in poultry muscle tissue with a sensitivity of 10 copies/μL was constructed using a set of commercial, fully automated nucleic acid analyzer with integrated detection based on magnetic bead method for nucleic acid extraction and PCR fluorescence detection. For 20 simulated samples, the integrated detection system was consistent with the results of qPCR experiments after conventional laboratory extraction, while the closed cassette-based detection reduced the chance of contamination occurrence, making the results more reliable and accurate, which is ideal for immediate on-site rapid detection.

Influence of Media Composition on the Level of Bovine Satellite Cell Proliferation.

It is predicted that already in 2040, 35% of requirements for meat will be provided by in vitro production. Recreating the course of myogenesis in vitro, and thus resembling a structure of muscle tissue, is the basis for research focusing on obtaining cultured meat and requires providing relevant factors supporting the proliferation of satellite cells-being precursors of skeletal muscles. The present work aimed to develop the composition of the medium that would most effectively stimulate the proliferation of bovine satellite cells (BSCs). The modeling and optimization methods included the measurements of the synergistic, co-stimulatory effect of three medium components: the amount of glucose, the type of serum (bovine or horse), and the amount of mitogenic factor-bFGF. Additionally, the qPCR analyses determined the expression of genes involved in myogenesis, such as Pax7 and Myogenic Regulatory Factors, depending on the level of the tested factor. The results showed significant positive effects of serum type (bovine serum) and mitogenic factor (addition of 10 ng/mL bFGF) on the proliferation rate. In turn, qPCR analysis displayed no significant differences in the relative expression level of Pax7 genes and MRF factors for both factors. However, a statistically higher Pax7 and Myf5 gene expression level was revealed when a low glucose medium was used (p < 0.05). In conclusion, the components of the medium, such as bovine serum and the addition of a mitogenic factor at the level of 10 ng/mL, ensure a higher proliferation rate of BSCs and lower glucose content ensured the expression of crucial genes in the self-renewal of the satellite cell population.

Hyperglycaemia Attenuated C2C12 Myoblast Proliferation and Induced Skeletal Muscle Atrophy via Modulating Myogenic Regulatory Factors Genes Expression in Diabetic Rats

Diabetes mellitus is characterised by high blood glucose level termed hyperglycaemia (HG). It has been reported to affect skeletal muscle by inducing skeletal muscle atrophy and skeletal muscle protein degradation leading to impairment of muscle function. This study aimed to investigate the effects of HG on the expression of myogenic regulatory factor genes in muscle progenitor cells and in skeletal muscle. The number of C2C12 myoblasts cultured in HG condition was significantly decreased compared to control in dose and time dependent manner. In addition, the number of Ki-67 positive nuclei was significantly decreased after treatment under HG condition. Real time PCR showed significant suppression of MyoD and myogenin, while Myf5 gene expression was significantly enhanced, compared to control. Furthermore, histological examinations of muscle fibres showed atrophy of tibialis anterior (TA) muscle from diabetic rats. The frequency of distribution of muscle fibre cross-sectional area (MCA) in diabetic rats was shifted leftward from that of normal control rats. In contrast, the MyoD and myogenin expression in TA muscle of diabetic rats were significantly increased compared to normal control rats. This study provides novel knowledge on the changing myogenic regulatory factor gene expression in hyperglycaemic condition, both in vitro and in vivo, leading to skeletal muscle atrophy.

Glutamine and leucine administration attenuates muscle atrophy in sepsis

AimsThis study investigated whether l-glutamine (Gln) and/or l-leucine (Leu) administration could attenuate muscle atrophy in a mouse model of cecal ligation and puncture (CLP)-induced sepsis. Materials and methodsSeptic mice were given a daily intraperitoneal injection of Gln, Leu, or Gln plus Leu, and mice were sacrificed on either day 1 or 4 after CLP. Blood and muscles were collected for analysis of amino acid contents and markers related to protein degradation, muscle regeneration, and protein synthesis. Key findingsLeu treatment alone increased both muscle mass and total muscle protein content on day 4 after CLP. Gln administration reduced muscular Gln contents on day 1 and enhanced plasma Gln levels on day 4. Higher plasma branched-chain amino acid (BCAA) abundances and lower muscular BCAA levels were observed in Leu-treated mice on day 4. Gln and Leu individually suppressed muscle expressions of the E3 ubiquitin ligase genes, Trim63 and Fbxo32, on day 4 after CLP. As to muscle expressions of myogenic genes, both Gln and Leu upregulated Myog expression on day 1, but Leu alone enhanced Myf5 gene expression, whereas Gln plus Leu increased MyoD and Myog expression levels on day 4. Akt/mammalian target of rapamycin (mTOR) signaling was only activated by Gln and Leu when individually administered. SignificanceGln and/or Leu administration reduces sepsis-induced muscle degradation and promotes myogenic gene expressions. Leu treatment alone had more-pronounced effects on maintaining muscle mass during sepsis. A combination of Gln and Leu failed to show synergistic effects on alleviating sepsis-induced muscle atrophy.

PSIX-34 Omega-3 fatty acids are more likely to cause C2C12 cell adipose deposition and decomposition compared with palmitic acid

Abstract It is usually considered that n-3 polyunsaturated fatty acids (n-3 PUFAs) to be beneficial to health and are widely provided as a dietary supplement during pregnancy. However, the risk of n-3 PUFAs overdosage on fetal development has rarely been studied. The objective of this study is to explore the effect of different concentrations of n-3 PUFAs [docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)] and a saturated fatty acid (palmitic acid) on the differentiation of C2C12 myoblasts. Oil Red O staining showed that n-3 PUFAs are more likely to cause adipogenesis. C2C12 cells were induced apoptosis when the concentration of EPA and DHA is 75µM, respectively. The expression of PPARγ gene expression in the EPA+DHA 50μM group was significantly increased, while in the palmitic acid treatment, the gene expression of AP2 (in the ≥10 μM groups) and C/EBPβ (50 μM) were significantly reduced (P &amp;lt; 0.05). Among the lipolysis related genes, HSL increased significantly in the EPA+DHA groups (≥ 25 μM), while MGL decreased significantly in both the EPA+DHA groups (≥ 10 μM) and palmitic acid group (≥ 100 μM) (P &amp;lt; 0.05). In the EPA+DHA groups, the expression of myogenic genes Mrf4 (≥10 μM), MyoD (≥50 μM), and MyoG (≥25 μM) were significantly reduced (P &amp;lt; 0.05). Gene expression of Myf5 decreased significantly when the concentration of palmitic acid was higher than 100 μM (P &amp;lt; 0.05). Among thermogenesis-related genes, in the EPA+DHA treatment, Prdm16 increased (≥50 μM), and UCP3 decreased significantly (≥50 μM)(P &amp;lt; 0.05). After palmitic acid administration, Prdm16 (≥50 μM) and UCP1 (≥100 μM) was significantly reduced, and UCP3 was significantly increased only in the highest concentration group (150 μM) (P &amp;lt; 0.05). In short, compared to palmitic acid, the treatment of EPA+DHA in the differentiation culture of C2C12 cells can enhance the deposition and decomposition of adipose in a lower concentration.

Graphene oxide nanofilm and the addition of l-glutamine can promote development of embryonic muscle cells

BackgroundFormation of muscular pseudo-tissue depends on muscle precursor cells, the extracellular matrix (ECM)-mimicking structure and factors stimulating cell differentiation. These three things cooperate and can create a tissue-like structure, however, their interrelationships are relatively unknown. The objective was to study the interaction between surface properties, culture medium composition and heterogeneous cell culture. We would like to demonstrate that changing the surface properties by coating with graphene oxide nanofilm (nGO) can affect cell behaviour and especially their need for the key amino acid l-glutamine (L-Glu).ResultsChicken embryo muscle cells and their precursors, cultured in vitro, were used as the experimental model. The mesenchymal stem cell, collected from the hind limb of the chicken embryo at day 8 were divided into 4 groups; the control group and groups treated with nGO, L-Glu and nGO supplied with L-Glu (nGOxL-Glu). The roughness of the surface of the plastic plate covered with nGO was much lower than a standard plate. The test of nGO biocompatibility demonstrated that the cells were willing to settle on the nGO without any toxic effects. Moreover, nGO by increasing hydrophilicity and reducing roughness and presumably through chemical bonds available on the GO surface stimulated the colonisation of primary stromal cells that promote embryonic satellite cells. The viability significantly increased in cells cultured on nGOxL-Glu. Observations of cell morphology showed that the most mature state of myogenesis was characteristic for the group nGOxL-Glu. This result was confirmed by increasing the expression of MYF5 genes at mRNA and protein levels. nGO also increased the expression of MYF5 and also very strongly the expression of PAX7 at mRNA and protein levels. However, when analysing the expression of PAX7, a positive link was observed between the nGO surface and the addition of L-Glu.ConclusionsThe use of nGO and L-Glu supplement may improve myogenesis and also the myogenic potential of myocytes and their precursors by promoting the formation of satellite cells. Studies have, for the first time, demonstrated positive cooperation between surface properties nGO and L-Glu supplementation to the culture medium regarding the myogenic potential of cells involved in muscle formation.

Muscle-specific gene expression and metabolic enzyme activities in Atlantic salmon Salmo salar L. fry reared under different photoperiod regimes.

This study was conducted to characterise the muscle-specific gene expression, energy metabolism level and growth rates of Atlantic salmon Salmo salar L. reared under different photoperiod regimes. The effects of two photoperiod regimes - LD 16:8 (16 h light:8 h dark) and LD 24:0 (24 h light:0 h dark) over a period of 3 months (August to October) on growth, energy metabolism enzyme activities (cytochrome c oxidase, COX; lactate dehydrogenase, LDH; and aldolase) and the gene expression levels of myogenic regulatory factors (MRFs - MyoD1 paralogues (MyoD1a, MyoD1b, MyoD1c), Myf5, MyoG), myostatin paralogues (MSTN-1a, MSTN-1b, MSTN-2a) and the fast skeletal myosin heavy chain (MyHC) in the muscles of Atlantic salmon underyearling fry (0+) were investigated. The experiment was conducted in a fish hatchery with natural variations in water temperature. The results were compared with those obtained in salmon reared under the lighting conditions of a fish hatchery (HL, hatchery lighting). The results revealed that the fry reared under constant light (LD 24:0) grew faster and were bigger at the end of the experiment. Fishes reared within the photoperiod regime LD 16:8 had a lower growth rate. COX activity was lower in fish under the LD 16:8 regime compared with the LD 24:0 group. The LDH and aldolase enzyme activities were higher in the group with constant light in comparison to control in the beginning of September. The expression level for all of the genes studied variated during the duration of the experiment, and MyHC, MyoG, MyoD1a and Myf5 expression depended on the light regime as well. The more noticeable changes in gene expression occurred in October. The MyHC and MyoG mRNA levels increased, accompanied by MyD1c gene expression, in both groups that had additional lighting (LD 16:8 and LD24:0) at the beginning of October and were higher than the HL group. In the HL group, the elevation of MyHC and MyoG mRNA was gradual during October, but there was a sharp increase in Myf5 expression at the beginning of October. MyoD1 paralogues differently expressed during the experiment. The MyoD1a mRNA level was elevated at the end of October along with MyHC and MyoG expression, but MyoD1b and MyoD1c mRNA levels decreased along with Myf5 gene expression. The expression of MSTN paralogues were elevated with increases in MyHC and MRFs transcripts. These findings show that constant light has a positive effect on the growth rate of salmon, affecting the aerobic and anaerobic capacity in their muscles. The alterations in muscle-specific gene expression between the groups with different light indicated that the mechanisms for regulating muscle growth processes in fish depend on photoperiod duration.

Isolation, in-vitro expansion, and characterization of human muscle satellite cells from the rectus abdominis muscle

Introduction: Satellite cells are a resident population of stem cells beneath the basal lamina of mature skeletal muscle fibers. Their capacity to regenerate muscle makes them a potentially ideal source for human cell therapy with respect to muscle-related diseases such as urinary and fecal incontinence, and others. In this study, we describe a protocol to isolate, expand in-vitro, and characterize human muscle satellite cells from the rectus abdominis muscle. Materials and Methods: Muscle biopsies from human donors were harvested, digested using collagenase type II, and then plated on extracellular matrix-coated plates. Results: Immunocytochemistry revealed that satellite cells on day 8 were 70–80% Pax7 positive; in contrast, cells expanded until day 12 showed 50–75% positivity for Pax7. The real-time polymerase chain reaction for day 8 culture indicated four-fold increase in Pax3 and Pax7 gene expression, four-fold increase in MyoD gene expression, and five-fold increase in Myf5 gene expression. Conclusion: These findings suggest that satellite cells can be cultured until day 8 for translational purposes. The protocol described here is modest, operational, and reproducible and involves only basic cell culture equipment.

Sex- and age-dependent expression of Pax7, Myf 5, MyoG, and Myostatin in yak skeletal muscles.

The aim of this study was to investigate the myogenic factor mRNA expression pattern of Pax7, Myf5, MyoG, and Myostatin mRNA at different ages, sexes, and muscle tissues of Datong yaks. The expression patterns in semimembranosus (SM), quadriceps femoris (QF), and triceps muscle of arm (TM) were detected by quantitative real-time polymerase chain reaction and compared using biostatistics. The results showed that the Pax7 gene expression levels were higher in the hindquarters (SM and QF) than in the forequarters, and was higher in male compared to in female muscle (P ≤ 0.05). The Myf5 gene expression levels of male yaks were highest in QF (P ≤ 0.05), whereas the expression levels of female yaks were highest in TM (P ≤ 0.05). Female MyoG gene expression levels were higher in QF and TM compared to in male yaks. The MyoG expression was higher in all muscles at 6 months old compared to in 3-year-old muscle. The highest MSTN gene expression was found in 6-month-old TM muscle and in QF muscle of 3 years (P ≥ 0.05). In conclusion, yak muscles showed different growth patterns depending on position. At 6 months of age, the satellite cells in the male hindquarter muscles and the female forequarter muscle showed a strong proliferative ability, at the same time the satellite cells in all female muscles had a powerful differentiation ability. Hindquarter muscles appear to mainly grow at younger ages and forequarters mainly grow at older ages.

Lentivirus-mediated RNA interference of myostatin gene affects MyoD and Myf5 gene expression in duck embryonic myoblasts

The aim of this study was to construct lentivirus-mediated short hairpin RNA (shRNA) vectors targeting the duck MSTN gene and investigate whether these vectors can affect the development of duck primary cultured embryonic myoblasts.MSTN mRNA levels in the myoblasts were detected using quantitative real-time polymerase chain reaction (PCR), cell proliferation was assessed by MTT assays and cell differentiation was assayed by photography.MSTN mRNA levels in PLL3.7-MSTN-shRNA1, PLL3.7-MSTN-shRNA2 and PLL3.7-MSTN-shRNA3 lentivirus-mediated shRNA groups were reduced by 61.6%, 76.9% and 79.1%, respectively, compared to control cells.Down-regulation of MSTN in duck embryonic myoblasts stimulated cell proliferation and inhibited differentiation, accompanied by a greater than twofold down-regulation of MyoD expression and up-regulation of Myf5 expression.These results revealed that silencing of MSTN changes the development of duck embryonic myoblasts by regulating the expression level of MyoD and Myf5 genes.

Proliferation rates of bovine primary muscle cells relate to liveweight and carcase weight in cattle.

Muscling in cattle is largely influenced by genetic background, ultimately affecting beef yield and is of major interest to the beef industry. This investigation aimed to determine whether primary skeletal muscle cells isolated from different breeds of cattle with a varying genetic potential for muscling differ in their myogenic proliferative capacity. Primary skeletal muscle cells were isolated and cultured from the Longissimus muscle (LM) of 6 month old Angus, Hereford and Wagyu X Angus cattle. Cells were assessed for rate of proliferation and gene expression of PAX7, MYOD, MYF5, and MYOG. Proliferation rates were found to differ between breeds of cattle whereby myoblasts from Angus cattle were found to proliferate at a greater rate than those of Hereford and Wagyu X Angus during early stages of growth (5–20 hours in culture) in vitro (P < 0.05). The proliferation rates of myoblasts during early stages of culture in vitro were also found to be positively related to the liveweight and carcase weight of cattle (P < 0.05). Gene expression of MYF5 was also found to be significantly down-regulated in WagyuX compared with Angus cattle (P < 0.05). These findings suggest that early events during myogenesis are important for determining liveweight and caracase weights in cattle.

Cloning and Expression Profiles of Myf5 Gene of Yak

To reveal the sequence characteristic and expression pattern of Myf5 gene in Jiulong yaks (Bos grunniens), a full-length cDNA of Myf5 was cloned from yak muscle tisssue by RT-PCR. The cDNA obtained was 821bp nucleotide (nt) long with an ORF of 768 bp which encoding 255 amino acids. Compared with cattle, sheep, pig, horse, human, pygmy chimpanzee, mouse, rat and dog, the homology of amino acid sequences were higher (89- 9%), but lower in Zebrafish (60%). SQ RT-PCR analysis showed that Myf5 gene expression was observed only in longissimus muscle, but not be detected in heart, liver, kidney, spleen and adipose tissues. The expression level of Myf5 gene in longissium muscle of 0.5 and over 9 years old yaks was significantly higher than those of 3.5-5.5 years old yaks (p<0.05). These results suggest that Myf5 may play an important role in the regulation of muscle growth and development of yak.

Direct molecular regulation of the myogenic determination gene Myf5 by Pax3, with modulation by Six1/4 factors, is exemplified by the −111 kb-Myf5 enhancer

The Myf5 gene plays an important role in myogenic determination during mouse embryo development. Multiple genomic regions of the Mrf4–Myf5 locus have been characterised as enhancer sequences responsible for the complex spatiotemporal expression of the Myf5 gene at the onset of myogenesis. These include an enhancer sequence, located at −111kb upstream of the Myf5 transcription start site, which is responsible of Myf5 activation in ventral somitic domains (Ribas et al., 2011. Dev. Biol. 355, 372–380). We show that the −111kb-Myf5 enhancer also directs transgene expression in some limb muscles, and is active at foetal as well as embryonic stages. We have carried out further characterisation of the regulation of this enhancer and show that the paired-box Pax3 transcription factor binds to it in vitro as in vivo, and that Pax binding sites are essential for its activity. This requirement is independent of the previously reported regulation by TEAD transcription factors. Six1/4 which, like Pax3, are important upstream regulators of myogenesis, also bind in vivo to sites in the −111kb-Myf5 enhancer and modulate its activity. The −111kb-Myf5 enhancer therefore shares common functional characteristics with another Myf5 regulatory sequence, the hypaxial and limb 145bp-Myf5 enhancer, both being directly regulated in vivo by Pax3 and Six1/4 proteins. However, in the case of the −111kb-Myf5 enhancer, Six has less effect and we conclude that Pax regulation plays a major role in controlling this aspect of the Myf5 gene expression at the onset of myogenesis in the embryo.

Regulation of Myf5 Early Enhancer by Histone Acetyltransferase p300 during Stem Cell Differentiation.

Skeletal myogenesis is an intricate process coordinated temporally by multiple myogenic regulatory factors (MRF) including Myf5, which is the first MRF expressed and marks the commitment of skeletal muscle lineage. The expression of Myf5 gene during early embryogenesis is controlled by a set of enhancer elements, and requires the histone acetyltransferase (HAT) activity of transcriptional coactivator p300. However, it is unclear as to how different regulatory signals converge at enhancer elements to regulate early Myf5 gene expression, and if p300 is directly involved. We show here that p300 associates with the Myf5 early enhancer at the early stage of stem cell differentiation, and its HAT activity is important for the recruitment of β-catenin to this early enhancer. In addition, histone H3-K27 acetylation, but not H3-K9/14, is intimately connected to the p300 HAT activity. Thus, p300 is directly involved in the regulation of the Myf5 early enhancer, and is important for specific histone acetylation and transcription factor recruitment. This connection of p300 HAT activity with H3-K27 acetylation and β-catenin signalling during myogenic differentiation in vitro offers a molecular insight into the enhancer-elements participation observed in embryonic development. In addition, pluripotent stem cell differentiation is a valuable system to dissect the signal-dependent regulation of specific enhancer element during cell fate determinations.

Effect of the g.–723G→T Polymorphism in the Bovine Myogenic Factor 5 (Myf5) Gene Promoter Region on Gene Transcript Level in the Longissimus Dorsi Muscle and on Meat Traits of Polish Holstein-Friesian Cattle

Myogenic factor 5 (Myf5), a product of the Myf5 gene, belongs to the MRF family of basic helix-loop-helix transcription factors that regulate myogenesis. Their roles in muscle growth and development make their genes candidates for molecular markers of meat production in livestock, but nucleotide sequence polymorphism has not been thoroughly studied in MRF genes. We detected four single nucleotide polymorphisms (SNPs) within exon 1 of the Myf5 gene, encoding the NH-terminal transactivation domain of the Myf5 protein. Three of these mutations change the amino acid sequence. The distribution of these SNPs was highly skewed in cattle populations; most of the mutations were found in only a few or even single individuals. Of the nine SNPs found in the promoter region of Myf5, one (transversion g.-723G-->T) was represented by all three genotypes distributed in the cattle populations studied. This polymorphism showed an influence on Myf5 gene expression in the longissimus dorsi muscle and was associated with sirloin weight and fat weight in sirloin in carcasses of Holstein-Friesian cattle.

The regulatory mechanisms that underlie inappropriate transcription of the myogenic determination gene Myf5 in the central nervous system

Myf5 is a key myogenic determination factor, specifically present at sites of myogenesis. Surprisingly, during mouse development, this gene is also transcribed in restricted areas of the central nervous system, although the Myf5 protein is not detectable. We have investigated the regulation of Myf5 expression in the central nervous system. Using both in ovo electroporation in the chick embryo and transgenesis in the mouse, we show that regulatory sequences that direct neuronal Myf5 transcription are present in a distal element located between −55 and −54.3 Kb from the Myf5 gene. An Oct6/Tst1 binding site is required for embryonic brain expression, and in the Oct6 mutant mouse embryo, Myf5 transcripts are no longer detectable in the brain. The Wnt-β catenin signalling pathway is also implicated. Finally we show that post-transcriptional regulation of Myf5 gene expression involves miRNA repression acting through the Myf5-3′UTR.

The early epaxial enhancer is essential for the initial expression of the skeletal muscle determination gene Myf5 but not for subsequent, multiple phases of somitic myogenesis.

Vertebrate myogenesis is controlled by four transcription factors known as the myogenic regulatory factors (MRFs): Myf5, Mrf4, myogenin and MyoD. During mouse development Myf5 is the first MRF to be expressed and it acts by integrating multiple developmental signals to initiate myogenesis. Numerous discrete regulatory elements are involved in the activation and maintenance of Myf5 gene expression in the various muscle precursor populations, reflecting the diversity of the signals that control myogenesis. Here we focus on the enhancer that recapitulates the first phase of Myf5 expression in the epaxial domain of the somite, in order to identify the subset of cells that first transcribes the gene and therefore gain insight into molecular, cellular and anatomical facets of early myogenesis. Deletion of this enhancer from a YAC reporter construct that recapitulates the Myf5 expression pattern demonstrates that this regulatory element is necessary for expression in the early epaxial somite but in no other site of myogenesis. Importantly, Myf5 is subsequently expressed in the epaxial myotome under the control of other elements located far upstream of the gene. Our data suggest that the inductive signals that control Myf5 expression switch rapidly from those that impinge on the early epaxial enhancer to those that impinge on the other enhancers that act later in the epaxial somite, indicating that there are significant changes in either the signalling environment or the responsiveness of the cells along the rostrocaudal axis. We propose that the first phase of Myf5 epaxial expression, driven by the early epaxial enhancer in the dermomyotome, is necessary for early myotome formation, while the subsequent phases are associated with cytodifferentiation within the myotome.

Zygotic Wnt/β-Catenin Signaling Preferentially Regulates the Expression of Myf5 Gene in the Mesoderm of Xenopus

Zygotic Wnt signaling has been shown to be involved in dorsoventral mesodermal patterning in Xenopus embryos, but how it regulates different myogenic gene expression in the lateral mesodermal domains is not clear. Here, we use transient exposure of embryos or explants to lithium, which mimics Wnt/β-catenin signaling, as a tool to regulate the activation of this pathway at different times and places during early development. We show that activation of Wnt/β-catenin signaling at the early gastrula stage rapidly induces ectopic expression of XMyf5 in both the dorsal and ventral mesoderm. In situ hybridization analysis reveals that the induction of ectopic XMyf5 expression in the dorsal mesoderm occurs within 45 min and is not blocked by the protein synthesis inhibitor cycloheximide. By contrast, the induction of XMyoD is observed after 2 h of lithium treatment and the normal expression pattern of XMyoD is blocked by cycloheximide. Analysis by RT-PCR of ectodermal explants isolated soon after midblastula transition indicates that lithium also specifically induces XMyf5 expression, which takes place 30 min following lithium treatment and is not blocked by cycloheximide, arguing strongly for an immediate-early response. In the early gastrula, inhibition of Wnt/β-catenin signaling blocks the expression of XMyf5 and XMyoD, but not of Xbra. We further show that zygotic Wnt/β-catenin signaling interacts specifically with bFGF and eFGF to promote XMyf5 expression in ectodermal cells. These results suggest that Wnt/β-catenin pathway is required for regulating myogenic gene expression in the presumptive mesoderm. In particular, it may directly activate the expression of the XMyf5 gene in the muscle precursor cells.