Biomedical subjects

E Bober

Publications and source records attributed to E Bober.

At least 37 records · Page 2Linked to original sources

PubMed1994

Initial steps of myogenesis in somites are independent of influence from axial structures.

Formation of paraxial muscles in vertebrate embryos depends upon interactions between early somites and the neural tube and notochord. Removal of both axial structures results in a complete loss of epaxial myotomal muscle, whereas hypaxial and limb muscles develop normally. We report that chicken embryos, after surgical removal of the neural tube at the level of the unsegmented paraxial mesoderm, start to develop myotomal cells that express transcripts for the muscle-specific regulators MyoD and myogenin. These cells also make desmin, indicating that the initial steps of axial skeletal muscle formation can occur in the absence of the neural tube. However, a few days following the extirpation, the expression of MyoD and myogenin transcripts gradually disappears, and becomes almost undetectable after 4 days. From these observations we conclude that the neural tube is not required for the generation of the skeletal muscle cell lineage, but may support the survival or maitenance of further differentiation of the myotomal cell compartment. Notochord transplanted medially or laterally to the unsegmented paraxial mesoderm leads to a ventralization of axial structures but does not entirely prevent the early appearance of myoblasts expressing MyoD transcripts. However, the additional notochord inhibits subsequent development and maturation of myotomes. Taken together, our data suggest that neural tube promotes, and notochord inhibits, the process of myogenesis in axial muscles at a developmental step following the initial expression of myogenic bHLH regulators.

Animals↗

PubMed1994

MyoD expression marks the onset of skeletal myogenesis in Myf-5 mutant mice.

The expression pattern of myogenic regulatory factors and myotome-specific contractile proteins was studied during embryonic development of Myf-5 mutant mice by in situ hybridization and immunohistochemistry. In contrast to somites in wild-type embryos, no expression of myogenin and Myf-6 (MRF4), or any other myotomal markers was detected in mutant animals at E9.0 and E10.0 indicating that Myf-5 plays a crucial role during this developmental period. Significantly, the onset of MyoD expression in rostral somites of E10.5 embryos was unaffected by the Myf-5 mutation suggesting that the activation of the MyoD gene occurs independently of Myf-5 at the correct developmental time. Immediately after the activation of MyoD myogenin transcripts and protein accumulated within the myotome. The first contractile proteins of the sarcomeric apparatus appeared slightly later. By E11.5 the expression of muscle markers were indistinguishable between wild-type and Myf-5 mutant mice. The migration of muscle precursor cells that leave the somites to form limb musculature was monitored in Myf-5-mutant mice by Pax-3 expression. Pax-3-positive cells were equally found in somites and limbs of E10.0 wild-type and mutant mice indicating that myogenic factor expression at the level of somites is not a prerequisite for determination and subsequent migration of limb precursor cells.

Animals↗

PubMed1994

Pax-3 is required for the development of limb muscles: a possible role for the migration of dermomyotomal muscle progenitor cells.

Limb muscles in vertebrates originate from dermomyotomal cells, which during early development migrate from the ventrolateral region of somites into the limb buds. These progenitor cells do not express any muscle-specific marker genes or myogenic transcription factors until they reach their destination in the limbs. Here, we demonstrate by in situ hybridization that myogenic cells in somites and a population of presumably migratory muscle precursor cells in somatopleural tissue as well as myoblasts in the developing limbs express Pax-3. Significantly, in homozygous splotch mutant mice, which synthesize altered Pax-3 mRNA but make no normal protein, no cells positive for Pax-3 transcripts can be detected in the region of migrating limb muscle precursors or in the limb itself. In contrast, myotomal precursor cells and axial skeletal muscles contain Pax-3 transcripts also in the mutant. Interestingly, these animals fail to develop limb musculature as demonstrated by the lack of hybridization with various probes for myogenic transcription factors (Myf-5, myogenin, MyoD) but make apparently normal axial muscles. These observations suggest that Pax-3 is necessary for the formation of limb muscles, affecting either the generation of myogenic precursors in the somitic dermomyotome or the migration of these cells into the limb field.

Animals↗

PubMed1992

Inhibition of muscle differentiation by the adenovirus E1a protein: repression of the transcriptional activating function of the HLH protein Myf-5.

Myogenic differentiation can be inhibited by the adenovirus E1a protein in the rat L6 muscle cell line. The present investigation provides evidence that E1a interferes with the expression of myogenin and the activity of Myf-5, the two myogenic helix-loop-helix (HLH) proteins that are expressed in L6 muscle cells. In nuclei of E1a-expressing L6 cells, Myf-5 protein accumulates to normal or even elevated levels and shows no alterations of its ability to bind to the DNA-binding site (CANNTG). However, trans-activation of muscle-specific reporter genes by Myf-5 is strongly inhibited. The same inhibition by E1a can be shown for the other myogenic HLH proteins, MyoD, myogenin, and MRF4/Myf-6, that have been expressed in 10T1/2 fibroblasts. In contrast to the normal level of Myf-5 expression, synthesis of myogenin is entirely abolished in the differentiation-defective L6-E1a cells. Here, we demonstrate that the carboxy-terminal trans-activator domain and probably the basic-HLH (bHLH) region of Myf-5 constitute targets for the inhibition by E1a. The effect of E1a depends on its intact transforming regions but not on the transcriptional activator domain. Our data suggest that activation of myogenin gene expression and the establishment of the differentiated phenotype may require functional Myf-5. Expression of the Myf-5 gene, however, is apparently independent of auto- or cross-regulation by the myogenic HLH proteins.

Adenovirus Early Proteins↗

PubMed1992

Regulation of myogenin expression in normal and transformed myogenic cell lines.

The control of myogenin (Myf-4), one of the muscle-specific regulatory proteins, is particularly interesting since its expression appears obligatory in myoblasts at the onset of differentiation. We isolated the human Myf-4 (myogenin) gene and determined promoter elements which direct cell type-specific expression and are subject to transactivation by the muscle transcription factors Myf-5 and MyoD1 in fibroblasts. Extrinsic signals such as serum components and purified growth factors or potential intracellular signals such as cAMP down-regulate transcription of the myogenin gene. Constitutive expression of the catalytic subunit of PKA completely suppresses transactivation of the myogenin promoter by Myf-5 or MyoD1 suggesting that cAMP may act via phosphorylation by PKA. In contrast to normal myogenic cell lines in which differentiation and myogenin expression can be induced by the removal of serum components, retinoic acid (RA) is required for differentiation in the rat rhabdomyosarcoma cell line BA-Han-1C. This model system was utilized to investigate factors which influence the balance between the transformed state and differentiation. Administration of retinoic acid to BA-Han-1C cells leads to the accumulation of myogenin mRNA approximately 48 h after the addition of RA. This late induction requires ongoing protein- and DNA-synthesis suggesting that trans- and cis-acting factors may be involved in the control. The critical involvement of myogenin in the process of terminal muscle differentiation was also demonstrated in the rat L6 muscle cell line which has been blocked for differentiation by the transforming protein E1a of Ad5 adenovirus. In cells which stably express E1a, myogenin expression is completely suppressed while Myf-5 continues to be synthesized normally. However, E1a inhibits the transactivator function of Myf-5, as demonstrated on GAL4-Myf5 chimeric proteins. A possible interpretation of this result is that Myf-5 or factors activated by Myf-5 are required for the expression of myogenin and myogenin itself is necessary for the terminal differentiation of myoblasts.

Blood↗

PubMed1991

Expression of members of the myf gene family in human rhabdomyosarcomas.

Northern analysis of tumour RNA has been used to examine the expression of members of the myf family of muscle determining genes (myf3, myf4, myf5 and myf6) in a series of 20 rhabdomyosarcomas. A 2.0 kb myf3 transcript was observed in 85% of tumours, a 1.8 kb myf4 transcript was detected in 70% of tumours and a 1.7 kb myf5 transcript was observed in 55% of tumours. Transcription of myf6 occurred in 28% of tumours, but there were several transcript sizes (1.2, 1.5, 2.0 and 3.5 kb) and in some individual tumours two or more transcripts were observed. Only two rhabdomyosarcomas, one classified as embryonal and one as pleomorphic, failed to exhibit transcription of members of the myf gene family. We were unable to detect transcription of myf genes in neuroblastomas, Wilms' tumours, hepatoblastomas, paediatric non-Hodgkin's lymphoma and leiomyosarcomas. When considered together these observations suggest that expression of myf genes could provide an extremely useful marker in the diagnosis of rhabdomyosarcoma.

Adolescent↗

PubMed1991

The muscle regulatory gene, Myf-6, has a biphasic pattern of expression during early mouse development.

The spatial and temporal expression pattern of the muscle regulatory gene Myf-6 (MRF4/herculin) has been investigated by in situ hybridization during embryonic and fetal mouse development. Here, we report that the Myf-6 gene shows a biphasic pattern of expression. Myf-6 transcripts are first detected in the most rostral somites of the mouse embryo at 9 d of gestation and accumulate progressively in myotomal cells along the rostro-caudal axis. This expression is transient and Myf-6 mRNA can no longer be detected in myotomal cells after day 12 post coitum (p.c.). In contrast to other muscle determination genes (MyoD1, myogenin, Myf-5), Myf-6 mRNA is not detected in limb buds or visceral arches and skeletal muscle of the mouse embryo (day 8-15 p.c.). In fetal mice, Myf-6 transcripts appear at day 16 p.c. in all skeletal muscles, and the gene continues to be expressed at a high level after birth. These results suggest that early Myf-6 expression may be restricted to a population of myogenic cells that does not contribute to the embryonic muscle masses in limb buds and visceral arches. The reappearance of Myf-6 mRNA in fetal skeletal muscle coincides approximately with secondary muscle fiber formation and the onset of innervation.

Base Sequence↗

PubMed1991

Early expression of the myogenic regulatory gene, myf-5, in precursor cells of skeletal muscle in the mouse embryo.

We have analysed by in situ hybridization the expression of myf-5, the murine homologue of the human myogenic regulatory sequence myf5, during embryogenesis in the mouse. myf-5 sequences were first detected in the earliest somites (from about 8 days p.c.) in the dermomyotome, before formation of the dermatome, myotome and sclerotome. The dermomyotome is classically considered to give rise to the precursor muscle cells of body and limb skeletal muscle. myf-5-positive cells were also detected early in the visceral arches and limb buds. In this case, as in somites, myf-5 expression precedes that of the two related myogenic regulatory sequences, myogenin and MyoD1, and indeed any other skeletal muscle marker examined to date. myf-5 is not detected at any stage in developing cardiac muscle. From 11.5 days p.c., the level of myf-5 transcripts begins to decrease to become undetectable (by in situ hybridization) from 14 days p.c. Both the appearance and disappearance of myf-5 follow the anteroposterior gradient of somite formation and maturation in the embryo. The time and place of myf-5 expression are consistent with a role in the early events of myogenic differentiation, possibly during determination of the myogenic lineage.

Animals↗

PubMed1991

Developmental patterns in the expression of Myf5, MyoD, myogenin, and MRF4 during myogenesis.

By using the polymerase chain reaction to amplify specific regions of the respective cDNAs, we have studied the expression of genes encoding the myogenic regulatory factors Myf5, MyoD, Myogenin, and MRF4 (Myf6, herculin) in cultured mouse muscle cells (inducible and permissive C2 cells and Sol8 cells). These cell lines may represent distinct stages in the progression of determined, or committed, muscle cells toward terminal differentiation. Transcripts for Myf5 were detected at the myoblast stage in all the committed muscle cells tested. Expression of the gene for MyoD appeared to be optional at the myoblast stage (MyoD is present in permissive myoblasts and absent from inducible myoblasts) but, like Myogenin, was found to accompany terminal differentiation. Furthermore, forced expression of MyoD converted inducible C2 cells into permissive cells. Expression of MRF4 was found to follow expression of the three other factors and to occur after the onset of terminal differentiation. Of particular interest was the finding that expression of MRF4 was temporally correlated with expression of the gene for the acetylcholine receptor epsilon-subunit that is characteristic of the adult receptor. In vivo, the only transcripts for myogenic regulatory factors to be detected in 8-day mouse embryos were those for Myf5, while expression of MRF4 followed expression of Myf5, MyoD, and Myogenin in developing limbs. Temporal and phenotypic differences related to the expression of Myf5, MyoD, Myogenin, and MRF4 suggest that these factors fulfil distinct roles in the control of myogenesis.

Animals↗

PubMed1990

A highly conserved enhancer downstream of the human MLC1/3 locus is a target for multiple myogenic determination factors.

A potent muscle-specific enhancer element, originally described in the rat myosin light chain (MLC) 1/3 locus located downstream of the coding region, is found in an analogous position in the human MLC1/3 gene. When linked to a CAT reporter gene and transfected into muscle or non-muscle cells, the human MLC enhancer directs high levels of muscle-specific gene expression from homologous or heterologous promoters, irrespective of position or orientation relative to the CAT transcription unit. A significant degree of sequence homology (over 85%) in the 3'-flanking regions of the two MLC genes is restricted to a 200 bp sequence which lies approximately 1.5 kb downstream of the polyadenylation site in both species. The human enhancer sequence includes binding sites for human myogenic determination factors containing a common basic helix-loop-helix motif, and it can be trans-activated to varying degrees in non-muscle cells by these factors. This study establishes the MLC enhancer as an evolutionarily conserved, integral component of the MLC1/3 locus which constitutes a novel target for the action of myogenic determination factors.

Animals↗

PubMed1990

Transcriptional activation domain of the muscle-specific gene-regulatory protein myf5.

The human muscle determination factor myf5, like MyoD and other members of the family of skeletal muscle-specific regulatory proteins, contains a highly conserved putative helix-loop-helix domain. In MyoD this motif is required for the initiation of myogenesis in C3H mouse 10T1/2 fibroblasts and other non-muscle cells as well as for transcriptional activation of muscle genes. High affinity DNA binding of MyoD to regulatory DNA elements in muscle genes requires the formation of heterodimers with ubiquitous helix-loop-helix proteins such as E12 or E47. To investigate the potential of myf5 as a transcription factor, we have fused the GAL4 DNA-binding domain to various parts of the myf5 protein and analysed the transactivation of a GAL4 reporter plasmid. Here we report that myf5 contains an intrinsic transcriptional activation domain which is distinct from the helix-loop-helix motif. The predominant transactivating effect is associated with the C-terminal half of the myf5 molecule. High-affinity sequence-specific DNA binding of myf5 also requires hetero-oligomeric association with the enhancer-binding protein E12 to confer muscle-specific transactivation.

Animals↗

PubMed1990

Identification of three developmentally controlled isoforms of human myosin heavy chains.

A set of cDNA clones coding for myosin heavy chains (MHC) was isolated from a human fetal skeletal muscle library. We have demonstrated by restriction mapping and nucleotide sequence analysis that the cDNAs represent three distinct transcripts, presumably the products of different genes. Furthermore, the pattern of mRNA expression indicates that the corresponding genes are regulated in a tissue-specific and developmental-stage-specific manner. While the cDNA clone gtMHC-V exhibits extensive sequence similarity to the rat beta-myosin heavy chain, the two other clones, gtMHC-F and gtMHC-E are very similar to the rat genes encoding the perinatal and embryonic myosin heavy chains, respectively. The mRNA corresponding to clone gt-MHC-V is highly expressed in heart and adult fast skeletal muscle and to a lesser extent in fetal skeletal muscle and adult slow skeletal muscle. The mRNAs corresponding to clones gtMHC-F and gtMHC-E are abundantly present in fetal skeletal muscle and are not present or barely detectable in heart and adult skeletal muscle.

Adult↗

PubMed1990

Myf-6, a new member of the human gene family of myogenic determination factors: evidence for a gene cluster on chromosome 12.

The Myf-6 gene, a novel member of the human gene family of muscle determination factors has been detected by its highly conserved sequence coding for a putative helix-loop-helix domain. This sequence motif is a common feature of all Myf factors and other regulatory proteins. The new Myf gene is located on human chromosome 12, approximately 6.5 Kb upstream of the Myf-5 locus in a closely linked cluster of myogenic determination genes. Myf-6 cDNAs were isolated from human and mouse skeletal muscle, the only tissue in which expression of the corresponding mRNA was observed. In contrast to human primary muscle cell cultures which express moderate levels of Myf-6 mRNA, most established rodent muscle cell lines completely lack this mRNA. Myogenic 10T1/2 cells, however, induced by the expression of either pEMSV-Myf-4 or pEMSV-Myf-5 activate their endogenous mouse Myf-6 gene. Constitutive expression of Myf-6 cDNA in C3H 10T1/2 fibroblasts establishes the muscle phenotype at a similar frequency to the previously characterized myogenic factors. Moreover, muscle-specific CAT reporter constructs containing either the human myosin light chain (MLC) enhancer or the promoter of the embryonic myosin light chain gene are activated in NIH 3T3 fibroblasts or in CV1 kidney cells by cotransfection of Myf-6 expression vehicles. This transcriptional activation occurs in the absence of any apparent conversion of the cellular phenotype of the recipient cells. Glutathione-S-transferase fusion proteins with Myf-3, Myf-4 or Myf-5 specifically bind to a MEF-like consensus sequence present in the human MLC enhancer and the MLC1 emb promoter. In contrast, the Myf-6 hybrid protein interacts weakly with the same sequences showing lower affinity and reduced specificity. Since co-expressed pEMSV-Myf-6, nevertheless, is able to activate transcription of the MLC-CAT reporter constructs in non-muscle tissue culture cells, the different DNA binding properties in vitro might suggest that transactivation of gene expression by Myf-6 involves distinct binding sites and/or additional protein factors.

Amino Acid Sequence↗

PubMed1990

Human embryonic/atrial myosin alkali light chain gene: characterization, sequence, and chromosomal location.

We have isolated and sequenced the gene encoding the human embryonic/atrial myosin alkali light chain isoform (MLC-1emb/A). The gene is split into seven exons by six introns; the last exon, as in all MLC isoform genes sequenced to date, is completely 3' untranslated sequence. Comparison of the MLC-1emb/A isoform gene with the other MLC-1 genes showed that the exon-intron arrangement of the human MLC-1emb/A isoform gene is analogous to that of the other MLC-1 type isoform genes. We have also mapped the human MLC-1emb/A isoform gene to the long arm of chromosome 17; the corresponding mouse gene has been mapped to chromosome 11. This gene, together with a number of others such as the collagen(I) alpha 1, galactokinase, and thymidine kinase genes, is part of the largest syntenic group between mouse and man.

Amino Acid Sequence↗

PubMed1989

Differential expression of myogenic determination genes in muscle cells: possible autoactivation by the Myf gene products.

The development of muscle cells involves the action of myogenic determination factors. In this report, we show that human skeletal muscle tissue contains, besides the previously described Myf-5, two additional factors Myf-3 and Myf-4 which represent the human homologues of the rodent proteins MyoD1 and myogenin. The genes encoding Myf-3, Myf-4 and Myf-5 are located on human chromosomes 11, 1, and 12 respectively. Constitutive expression of a single factor is sufficient to convert mouse C3H 10T1/2 fibroblasts to phenotypically normal muscle cells. The myogenic conversion of 10T1/2 fibroblasts results in the activation of the endogenous MyoD1 and Myf-4 (myogenin) genes. This observation suggests that the expression of Myf proteins leads to positive autoregulation of the members of the Myf gene family. Individual myogenic colonies derived from MCA C115 cells (10T1/2 fibroblast transformed by methylcholanthrene) express various levels of endogenous MyoD1 mRNA ranging from nearly zero to high levels. The Myf-5 gene was generally not activated in 10T1/2 derived myogenic cell lines but was expressed in some MCA myoblasts. In primary human muscle cells Myf-3 and Myf-4 mRNA but very little Myf-5 mRNA is expressed. In mouse C2 and P2 muscle cell lines MyoD1 is abundantly synthesized together with myogenin. In contrast, the rat muscle lines L8 and L6 and the mouse BC3H1 cells express primarily myogenin and low levels of Myf-5 but no MyoD1. Myf-4 (myogenin) mRNA is present in all muscle cell lines at the onset of differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1989

A novel human muscle factor related to but distinct from MyoD1 induces myogenic conversion in 10T1/2 fibroblasts.

We have isolated the cDNA encoding a novel human myogenic factor, Myf-5, by weak cross-hydridization to the mouse MyoD1 probe. Nucleotide sequence analysis and the identification of the corresponding gene indicate that human Myf-5 is a member of a small gene family which also contains the human homologue to MyoD1. Although structurally related to the mouse factor, the human Myf-5 constitutes a different protein which nevertheless is capable of inducing the myogenic phenotype in embryonic C3H mouse 10T1/2 'fibroblasts'. The existence of more than one MyoD1-like protein in human skeletal muscle is further suggested by the detection of several similar but distinct cDNA clones. The phenotypic conversion of 10T1/2 cells by the human factor is recognized by the capacity of the cells to form multinucleated syncytia and synthesize sarcomeric myosin heavy chains. Moreover, transient expression of Myf-5 in 10T1/2 cells leads to the activation of a co-transfected muscle-specific CAT reporter gene which by itself is transcriptionally silent in the non-muscle cell background. The deduced amino acid sequence of clone Myf-5 reveals a region which is highly similar to myc proteins and the developmental factors from Drosophila encoded by the achaete scute locus and the twist gene. The myc homology region and a preceding cluster of basic amino acids are located in a larger sequence domain with strong similarity to the mouse myogenic factor MyoD1. Two additional short segments with high serine and threonine content are conserved between the two proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1988

A novel human myosin alkali light chain is developmentally regulated. Expression in fetal cardiac and skeletal muscle and in adult atria.

We have isolated cDNA recombinant phages encoding the embryonic isoform of the myosin alkali light chain (MLC1emb) from a human fetal skeletal muscle library. The cDNA clones were detected by their weak cross-hybridization to a human MLC1F and MLC3F cDNA clone. Nucleotide sequence analysis of the complete cDNA (GT14) revealed an open reading frame for 197 amino acids. The derived protein sequence constitutes the first structural information on this myosin isoform of any organism. Remarkable structural similarities to other alkali MLC polypeptides, particularly to those of the slow-muscle type, are evident. Under conditions of high stringency, the GT14 clone hybridized to an abundant mRNA species in fetal ventricular muscle and adult atrial muscle, whereas in fetal skeletal muscle only a very weakly hybridizing mRNA component was detected. These mRNAs were indistinguishable by size and the thermal stability of their hybrids formed with the DNA insert of clone GT14. We therefore conclude that identical mRNA is expressed in these tissues, presumably transcribed from the same gene. According to its pattern of mRNA expression, the novel MLC isoform described here was designated as "embryonic and atrial myosin light chain" (MLC1emb/A) in reference to its developmental stage-specific and tissue-specific appearance in embryonic skeletal muscle, fetal ventricle and adult atrium.

Adult↗

PubMed1988

Alkali myosin light chains in man are encoded by a multigene family that includes the adult skeletal muscle, the embryonic or atrial, and nonsarcomeric isoforms.

A set of cDNA clones coding for alkali myosin light chains (AMLC) was isolated from fetal human skeletal muscle. Nucleotide sequence analysis and RNA expression patterns of individual clones revealed related sequences corresponding to (i) fast fiber type MLC1 and MLC3; (ii) the embryonic MLC that is also expressed in fetal ventricle and adult atrium (MLCemb); and (iii) a nonsarcomeric MLC isoform that is found in all nonmuscle cell types and smooth muscle. The AMLC gene family in man comprises unique copies for MLC1, MLC3 and MLCemb, and multiple copies for the nonsarcomeric MLC genes. The gene coding for MLC1 and MLC3 is located on human chromosome 2.

Amino Acid Sequence↗