Muscle development in Ciona intestinalis requires the b-HLH myogenic regulatory factor gene Ci-MRF

The activity of myogenic regulatory factor (MRF) genes is essential for vertebrate muscle development, whereas invertebrate muscle development is largely independent of MRF function. This difference indicates that myogenesis is controlled by distinct regulatory mechanisms in these two groups of animals. Here we used overexpression and gene knockdown to investigate the role in embryonic myogenesis of the single MRF gene of the invertebrate chordate Ciona intestinalis (Ci-MRF). Injection of Ci-MRF mRNA into eggs resulted in increased embryonic muscle-specific gene activity and revealed the myogenic activity of Ci-MRF by inducing the expression of four muscle marker genes, Acetylcholinesterase, Actin, Troponin I, and Myosin Light Chain in non-muscle lineages. Conversely, inhibiting Ci-MRF activity with antisense morpholinos down-regulated the expression of these genes. Consistent with the effects of morpholinos on muscle gene activity, larvae resulting from morpholino injection were paralyzed and their "muscle" cells lacked myofibrils. We conclude that Ci-MRF is required for larval tail muscle development and thus that an MRF-dependent myogenic regulatory network probably existed in the ancestor of tunicates and vertebrates. This possibility raises the question of whether the earliest myogenic regulatory networks were MRF-dependent or MRF-independent.     

Target gene selectivity of the myogenic basic helix-loop-helix transcription factor myogenin in embryonic muscle

The myogenic regulatory factors MyoD and myogenin are crucial for skeletal muscle development. Despite their importance, the mechanisms by which these factors selectively regulate different target genes are unclear. The purpose of the present investigation was to compare embryonic skeletal muscle from myogenin^+/+ and myogenin^−/− mice to identify genes whose expression was dependent on the presence of myogenin but not MyoD and to determine whether myogenin-binding sites could be found within regulatory regions of myogenin-dependent genes independent of MyoD. We identified a set of 140 muscle-expressed genes whose expression in embryonic tongue muscle of myogenin^−/− mice was downregulated in the absence of myogenin, but in the presence of MyoD. Myogenin bound within conserved regulatory regions of several of the downregulated genes, but MyoD bound only to a subset of these same regions, suggesting that many downregulated genes were selective targets of myogenin. The regulatory regions activated gene expression in cultured myoblasts and fibroblasts overexpressing myogenin or MyoD, indicating that expression from exogenously introduced DNA could not recapitulate the selectivity for myogenin observed in vivo. The results identify new target genes for myogenin and show that myogenin's target gene selectivity is not based solely on binding site sequences.     

A significant reduction of the diaphragm in mdx:MyoD-/-(9th) embryos suggests a role for MyoD in the diaphragm development

To further investigate the role of MyoD during skeletal myogenesis, we backcrossed mdx mutant mice (lacking dystrophin) with MyoD knock-out mice to obtain viable mice with MyoD allele on a pure mdx background. However, after nine generations of backcrossing, it was not possible to obtain a viable mdx:MyoD-/- phenotype (designated as: mdx:MyoD-/-(9th)). The compound-mutant embryos were examined just before birth. Essentially normal Myf5-dependent and most of the MyoD-dependent musculature was observed. By contrast, the skeletal muscle compartment of the diaphragm was significantly reduced. The mesenchymal compartment of the diaphragm was intact and no herniations were observed. Other examined organs (e.g., liver, kidney, brain, etc.) showed no histological abnormalities. Pulmonary hypoplasia was determined as the cause of neonatal death. Therefore, using a different approach, our new data supplement our previous findings and suggest an essential role for MyoD in development of skeletal muscle of the diaphragm. The failure of mdx:MyoD-/-(9th) diaphragm to develop normally is not caused by a reduced number of satellite cells, but from the inability of stem cells to progress through the myogenic program. Our data also suggest that functions of MyoD and Myf5 (and the respective muscle precursor cell sub-populations) are not entirely redundant by term, as previously suggested, since Myf5 is not capable of fully substituting for MyoD in the diaphragm development.     

Myo/Nog cell regulation of bone morphogenetic protein signaling in the blastocyst is essential for normal morphogenesis and striated muscle lineage specification

Cells that express MyoD mRNA, the G8 antigen and the bone morphogenetic protein (BMP) inhibitor noggin (Nog) are present in the epiblast before gastrulation. Ablation of “Myo/Nog” cells in the blastocyst results in an expansion of canonical BMP signaling and prevents the expression of noggin and follistatin before and after the onset of gastrulation. Once eliminated in the epiblast, they are neither replaced nor compensated for as development progresses. Older embryos lacking Myo/Nog cells exhibit severe axial malformations. Although Wnts and Sonic hedgehog are expressed in ablated embryos, skeletal muscle progenitors expressing Pax3 are missing in the somites. Pax3+ cells do emerge adjacent to Wnt3a+ cells in vitro; however, few undergo skeletal myogenesis. Ablation of Myo/Nog cells also results in ectopically placed cardiac progenitors and cardiomyocytes in the somites. Reintroduction of Myo/Nog cells into the epiblast of ablated embryos restores normal patterns of BMP signaling, morphogenesis and skeletal myogenesis, and inhibits the expression of cardiac markers in the somites. This study demonstrates that Myo/Nog cells are essential regulators of BMP signaling in the early epiblast and are indispensable for normal morphogenesis and striated muscle lineage specification.     

Identification of the Drosophila eIF4A gene as a target of the DREF transcription factor

The DNA replication-related element-binding factor (DREF) regulates cell proliferation-related gene expression in Drosophila. We have carried out a genetic screening, taking advantage of the rough eye phenotype of transgenic flies that express full-length DREF in the eye imaginal discs and identified the eukaryotic initiation factor 4A (eIF4A) gene as a dominant suppressor of the DREF-induced rough eye phenotype. The eIF4A gene was here found to carry three DRE sequences, DRE1 (-40 to -47), DRE2 (-48 to -55), and DRE3 (-267 to -274) in its promoter region, these all being important for the eIF4A gene promoter activity in cultured Drosophila Kc cells and in living flies. Knockdown of DREF in Drosophila S2 cells decreased the eIF4A mRNA level and the eIF4A gene promoter activity. Furthermore, specific binding of DREF to genomic regions containing DRE sequences was demonstrated by chromatin immunoprecipitation assays using anti-DREF antibodies. Band mobility shift assays using Kc cell nuclear extracts revealed that DREF could bind to DRE1 and DRE3 sequences in the eIF4A gene promoter in vitro, but not to the DRE2 sequence. The results suggest that the eIF4A gene is under the control of the DREF pathway and DREF is therefore involved in the regulation of protein synthesis.     

Myostatin and MyoD family expression in skeletal muscle of IGF-1 knockout mice

Insulin-like growth factor-1 (IGF-1) is a positive regulator in proliferation and differentiation of skeletal muscle cells, while myostatin (MSTN) is a member of transforming growth factor beta superfamily that acts as a negative regulator of skeletal muscle mass. The present study was performed to detail whether a correlation exists between MSTN and IGF-1 in skeletal muscle of IGF-1 knockout mice (IGF-1(-/-)) and their wild type (WT; i.e., IGF-1(+/+)) littermates. The body weight of IGF-1(-/-) animals was 32% that of WT littermates. The fiber cross-sectional areas (CSA) and number of fibers in M. rectus femoris of IGF-1(-/-) animals were 49 and 59% those of WT animals, respectively. Thus, muscle hypoplasia of IGF-1(-/-) undoubtedly was confirmed. Myostatin mRNA levels and protein levels were similar between M. gastrocnemius of IGF-1(-/-) and WT animals. Myostatin immunoreactivity was similarly localized in muscle fibers of both IGF-1(-/-) and WT M. rectus femoris. The mRNA levels of MyoD family (Myf5, MyoD, MRF4, myogenin) were differentially expressed in IGF-1(-/-)M. gastrocnemius, in which the mRNA expression of MRF4 and myogenin was significantly lower, whereas there were no changes in the mRNA expression of Myf5 and MyoD. These findings first describe that myostatin expression is not influenced by intrinsic failure of IGF-1, although MRF4 and myogenin are downregulated.     

Effects of one bout of endurance exercise on the expression of myogenin in human quadriceps muscle

The objective of this study was to investigate the cellular localisation of MyoD and myogenin in human skeletal muscle fibres as well as the possible alterations in the expression of MyoD and myogenin in response to a single bout of endurance exercise at 40% and 75% of maximum oxygen uptake (VO₂ max). Twenty-five biopsies (5 per subject) from the vastus lateralis muscle were obtained before exercise, from the exercising leg at 40% and 75% of VO₂ max and from the resting leg following these exercise bouts. The tyramide signal amplification-direct and the Vectastain ABC methods using specific monoclonal antibodies were used to determine the exact location of myogenin and MyoD, to identify muscle satellite cells and to determine myosin heavy chain (MyHC) composition. At rest, myonuclei did not express MyoD or myogenin. Following a single bout of exercise at 40% and 75% of VO₂ max, an accumulation of myogenin in myonuclei and not in satellite cells was observed in biopsies from the exercised leg but not in biopsies before exercise and from the resting leg. The number of myogenin-positive myonuclei varied among individuals indicating differences in the response to a single exercise bout. In conclusion, this immunohistochemical study showed that a rapid rearrangement of myogenin expression occurs in exercised human skeletal muscles in response to a single bout of exercise.     

Interferon regulatory factor 4 (IRF-4) targets IRF-5 to regulate Epstein-Barr virus transformation

The cellular interferon regulatory factor-4 (IRF-4), which is a member of IRF family, is involved in the development of multiple myeloma and Epstein-Barr virus (EBV)-mediated transformation of B lymphocytes. However, the molecular mechanism of IRF-4 in cellular transformation is unknown. We have found that knockdown of IRF-4 leads to high expression of IRF-5, a pro-apoptotic member in the IRF family. Overexpression of IRF-4 represses IRF-5 expression. Reduction of IRF-4 leads to growth inhibition, and the restoration of IRF-4 by exogenous plasmids correlates with the growth recovery and reduces IRF-5 expression. In addition, IRF-4 negatively regulates IRF-5 promoter reporter activities and binds to IRF-5 promoters in vivo and in vitro. Knockdown of IRF-5 rescues IRF-4 knockdown-mediated growth inhibition, and IRF-5 overexpression alone is sufficient to induce cellular growth inhibition of EBV-transformed cells. Therefore, IRF-5 is one of the targets of IRF-4, and IRF-4 regulates the growth of EBV-transformed cells partially through IRF-5. This work provides insight on how IRFs interact with one another to participate in viral pathogenesis and transformation.     

Two proteins act as the IUF1 insulin gene enhancer binding factor

IUF1 is a pancreatic β cell-specific factor which binds to the sequence 5′-CPyCTAATG-3′ (CT box) within the human insulin gene enhancer. Here we show that IUF1 is composed of 2 binding activities that can be separated by DEAE ion exchange chromatography. South Western blot analysis indicates that these distinct binding activities have apparent molecular weights of 115 kDa and 46 kDa.     

Identification of a critical control element directing expression of the muscle-specific transcription factor MRF4 in the mouse embryo

Skeletal muscle development in the vertebrate embryo critically depends on the myogenic regulatory factors (MRFs) including MRF4 and Myf5. Both genes exhibit distinct expression patterns during mouse embryogenesis, although they are genetically closely linked with multiple regulatory elements dispersed throughout the common gene locus. MRF4 has a biphasic expression profile, first in somites and later in foetal skeletal muscles. Here, we demonstrate by transgenic analysis that elements within a 7.5-kb promoter fragment of the MRF4 gene are sufficient to drive the embryonic wave of expression very similar to the endogenous gene in somites of mouse embryos. In contrast, a 3-kb fragment of the proximal promoter fails to support expression in the myotome, suggesting that essential cis-acting elements are located between -7.5 and -3 kb upstream of MRF4. Further analysis of this sequence delimits an essential region between -6.6 and -5.6 kb that together with the 3-kb promoter fragment directs transgene expression in the epaxial myotome of all somites during the appropriate developmental period. These data provide evidence that the partly overlapping expression patterns of Mrf4 and Myf5 in somites are controlled by distinct regulatory elements. We also show that 11.4 kb sequence upstream of MRF4, including the promoter and the somitic control region identified in this study, is not sufficient to elicit target specificity towards the strong Myf5 (-58/-48 kb) enhancer, suggesting that additional yet unidentified elements are necessary to convey promoter selectivity and protect the MRF4 gene from this enhancer.