Search PubMed⌕ Search

Biomedical subjects

S Tajbakhsh

Publications and source records attributed to S Tajbakhsh.

32 records · Page 2Linked to original sources
PubMed1996

Gene targeting the myf-5 locus with nlacZ reveals expression of this myogenic factor in mature skeletal muscle fibres as well as early embryonic muscle.

We have introduced the nlacZ reporter gene into the locus of the myogenic factor gene myf-5 by homologous recombination in embryonic stem (ES) cells. Targeted ES clones were injected into precompaction morula, and the beta-galactosidase expression pattern was monitored. These mice permit the sensitive visualization of myf-5 expression throughout the embryo, and provide a standard for comparing it with that seen with different myf-5/nlacZ transgenes. Thus, in a comparison using ES cells in chimaeric embryos containing the targeted or randomly integrated myf-5/nlacZ construct, we demonstrate that 5.5 kbp of myf-5 upstream flanking sequence including exon1 and most of intron1 directs some skeletal muscle expression, but this is neither qualitatively nor quantitatively equivalent to that of the endogenous gene. Myf-5 is expressed early, before terminal myogenesis takes place in the medial half of the somite, and subsequently it is a major myogenic factor as skeletal muscle forms. All skeletal muscle shows beta-galactosidase activity, even after birth, indicating that myf-5 expression is not confined to primary myotubes, which are derived from embryonic myoblasts, but is also present in muscles containing different adult fibre types. The presence of myf-5 transcripts from the endogenous gene in older muscle was confirmed by in situ hybridization. These results suggest that the myf-5 gene is not activated in only a subset of muscle cells and are consistent with the results on the MyoD knockout mice.

Animals↗
PubMed1996

How is myogenesis initiated in the embryo?

Skeletal myoblasts are derived from paraxial mesoderm, but how myoblasts acquire their identity is still a matter of speculation. The characterization of molecular markers and, in some cases, the analysis of mutations in the corresponding genes, has now made it possible to ask specific questions about this process. Specification of somite cell fate depends on epigenetic factors. Adjacent tissues, such as the neural tube, notochord, dorsal ectoderm and lateral mesoderm, act either positively or negatively on the different myogenic precursor populations in the somite. Candidate molecules for this complex signalling activity include sonic hedgehog and the Wnt proteins as positive signals, and BMP4 as a possible inhibitor. Although it is generally assumed that induction is required, some observations suggest that embryonic cells might have a tendency to undergo myogenesis as a 'default' pathway. By analogy with Drosophila, where the neurogenic genes affect myogenesis, the vertebrate homologues of notch and its ligands could be candidate molecules for a repression or derepression mechanism. Similar studies with cultured muscle cells also implicate other HLH factors as potential inhibitors of the MyoD family and, hence, of inappropriate myogenesis.

Animals↗
PubMed1996

Activation of different myogenic pathways: myf-5 is induced by the neural tube and MyoD by the dorsal ectoderm in mouse paraxial mesoderm.

Newly formed somites or unsegmented paraxial mesoderm (UPM) have been cultured either in isolation or with adjacent structures to investigate the influence of these tissues on myogenic differentiation in mammals. The extent of differentiation was easily and accurately quantified by counting the number of beta-galactosidase-positive cells, since mesodermal tissues had been isolated from transgenic mice that carry the n-lacZ gene under the transcriptional control of a myosin light chain promoter, restricting expression to striated muscle. The results obtained showed that axial structures are necessary to promote differentiation of paraxial mesoderm, in agreement with previous observations. However, it also appeared that the influence of axial structures could be replaced by dorsolateral tissues, adjacent to the paraxial mesoderm. To elucidate which of these tissues exerts this positive effect, we cultured the paraxial mesoderm with a variety of adjacent structures, either adherent to the mesoderm or recombined in vitro. The results of these experiments indicated that the dorsal ectoderm exerts a positive influence on myogenesis but only if left in physical proximity to it. In contrast, lateral mesoderm delays the positive effect of the ectoderm (and has no effect on its own) suggesting that this tissue produces an inhibitory signal. To investigate whether axial structures and dorsal ectoderm induce myogenesis through common or separate pathways, we dissected the medial half of the unsegmented paraxial mesoderm and cultured it with the adjacent neural tube. We also cultured the lateral half of the unsegmented paraxial mesoderm with adjacent ectoderm. The induction of the myogenic regulatory factors myf-5 and MyoD was monitored by double staining of cultured cells with antibodies against MyoD and beta-galactosidase since the tissues were isolated from mouse embryos that carry n-lacZ targeted to the myf-5 gene, so that myf-5 expressing cells could be easily identified by either histochemical or immunocytochemical staining for beta-galactosidase. After 1 day in culture myogenic cells from the medial half expressed myf-5 but not MyoD, while myogenic cells from the lateral half expressed MyoD but not myf-5. By the next day in vitro, however, most myogenic cells expressed both gene products. These data suggest that the neural tube activates myogenesis in the medial half of paraxial mesoderm through a myf-5-dependent pathway, while the dorsal ectoderm activates myogenesis through a MyoD-dependent pathway. The possible developmental significance of these observations is discussed and a model of myogenic determination in mammals is proposed.

Animals↗
PubMed1995

Myosin light chain 3F regulatory sequences confer regionalized cardiac and skeletal muscle expression in transgenic mice.

The myosin light chain IF/3F locus contains two independent promoters, MLC1F and MLC3F, which are differentially activated during skeletal muscle development. Transcription at this locus is regulated by a 3' skeletal muscle enhancer element, which directs correct temporal and tissue-specific expression from the MLC1F promoter in transgenic mice. To investigate the role of this enhancer in regulation of the MLC3F promoter in vivo, we have analyzed reporter gene expression in transgenic mice containing lacZ under transcriptional control of the mouse MLC3F promoter and 3' enhancer element. Our results show that these regulatory elements direct strong expression of lacZ in skeletal muscle; the transgene, however, is activated 4-5 d before the endogenous MLC3F promoter, at the time of initiation of MLC1F transcription. In adult mice, transgene activity is downregulated in muscles that have reduced contributions of type IIB fibers (soleus and diaphragm). The rostrocaudal positional gradient of transgene expression documented for MLC1F transgenic mice (Donoghue, M., J. P. Merlie, N. Rosenthal, and J. R. Sanes. 1991. Proc. Natl. Acad. Sci. USA. 88:5847-5851) is not seen in MLC3F transgenic mice. Although MLC3F was previously thought to be restricted to skeletal striated muscle, the MLC3F-lacZ transgene is expressed in cardiac muscle from 7.5 d of development in a spatially restricted manner in the atria and left ventricular compartments, suggesting that transcriptional differences exist between cardiomyocytes in left and right compartments of the heart. We show here that transgene-directed expression of the MLC3F promoter reflects low level expression of endogenous MLC3F transcripts in the mouse heart.

Animals↗
PubMed1995

Lineage restriction of the myogenic conversion factor myf-5 in the brain.

myf-5 is one of four transcription factors belonging to the MyoD family that play key roles in skeletal muscle determination and differentiation. We have shown earlier by gene targeting nlacZ into the murine myf-5 locus that myf-5 expression in the developing mouse embryo is closely associated with the restriction of precursor muscle cells to the myogenic lineage. We now identify unexpected expression of this myogenic factor in subdomains of the brain. myf-5 expression begins to be detected at embryonic day 8 (E8) in the mesencephalon and coincides with the appearance of the first differentiated neurons; expression in the secondary prosencephalon initiates at E10 and is confined to the ventral domain of prosomere p4, later becoming restricted to the posterior hypothalamus. This expression is observed throughout embryogenesis. No other member of the MyoD family is detected in these regions, consistent with the lack of myogenic conversion. Furthermore, embryonic stem cells expressing the myf-5/nlacZ allele yield both skeletal muscle and neuronal cells when differentiated in vitro. These observations raise questions about the role of myf-5 in neurogenesis as well as myogenesis, and introduce a new lineage marker for the developing brain.

Animals↗
PubMed1994

Mouse limb muscle is determined in the absence of the earliest myogenic factor myf-5.

myf-5 is the only member of the MyoD family of myogenic regulatory genes to be expressed in the mouse embryo prior to muscle cell differentiation. We have used the developing limb as a model in which to follow the formation of peripheral muscle, to address the question of whether myogenic precursor cells are already present in the limb bud before expression of myf-5. The lacZ reporter gene has been introduced into the myf-5 gene by homologous recombination so that its expression is under the control of the endogenous myf-5 locus. beta-Galactosidase (beta-gal) coloration provides a sensitive assay for myf-5+ cells. Embryos were generated from embryonic stem cells carrying this mutation, and the appearance of beta-gal+ (myf-5+) cells was followed during limb development in vivo. Limb buds, at a stage when they are beta-gal-, were cultured in vitro. After several days, beta-gal+ cells accumulated in the premuscle mass. We conclude that determined muscle precursor cells in the limb bud do not initially express any member of the MyoD family. Furthermore, myogenic precursor cells in the somite, which, according to the avian model, migrate from the ventral/lateral edge of the dermomyotome to form peripheral muscle masses, are also negative for these factors.

Animals↗
PubMed1994

A population of myogenic cells derived from the mouse neural tube.

Embryonic mouse neural tubes produce a variety of terminally differentiated cells in vitro, mostly neurons and glia. We report here that some of these cells differentiate into skeletal muscle cells. The possibility of mesoderm contamination was ruled out as follows. First, Dil+ muscle cells were present in cultures from a Dil-labeled neuroepithelium. Second, a small fraction of cultured neural tube cells coexpressed muscle myosin and neuronal beta III tubulin within the same cell. Third, embryos generated from embryonic stem cells in which nlacZ was targeted into the myogenic gene myf-5 expressed nlacZ in a localized region of the neural tube. These myf-5+ cells coexpress neuronal and muscle markers in culture. The developmental significance of this phenomenon is discussed in the context of overlapping regulatory networks between myogenesis and neurogenesis.

Animals↗
PubMed1993

Expression of myogenic factors in the mouse: myf-5, the first member of the MyoD gene family to be transcribed during skeletal myogenesis.

There are four myogenic factors of the MyoD family present in mammals, each have distinct patterns of expression during skeletal myogenesis in the mouse. This differs between axial (myotome) and peripheral (limb) muscle masses, which are derived from the medial and lateral parts of the somite, respectively. The onset of expression of the muscle structural genes also differs, suggesting differences in their activation threshold in response to different factors. In all cases myf-5 is the first gene to be expressed. In order to examine this more closely, we have introduced an nlsLacZ sequence into the myf-5 gene by homologous recombination. This has permitted us to follow expression of this gene at the cellular level. While the immediate precursors of the myotome are labelled, we have shown that peripheral muscle masses such as those in the limb are founded by muscle precursor cells which are initially myf-5 negative. At later stages of development the gene is transcribed in all skeletal muscles, irrespective of their origin and fibre type. We discuss the unexpected finding which was made with the myf-5/nlsLacZ mice, that myf-5 is also expressed in the central nervous system.

Animals↗
PubMed1992

Adenovirus as an expression vector in muscle cells in vivo.

Attempting gene transfer in muscle raises difficult problems: the nuclei of mature muscle fibers do not undergo division, thus excluding strategies involving replicative integration of exogenous DNA. As adenovirus has been reported to be an efficient vector for the transfer of an enzyme encoding gene in mice, we decided to explore its potential for muscle cells. Advantages of adenovirus vectors are their independence of host cell replication, broad host range, and potential capacity for large foreign DNA inserts. We constructed a recombinant adenovirus containing the beta-galactosidase reporter gene under the control of muscle-specific regulatory sequences. This recombinant virus was able to direct expression of the beta-galactosidase in myotubes in vitro. We report its in vivo expression in mouse muscles up to 75 days after infection. The efficiency and stability of expression we obtained compare very favorably with other strategies proposed for gene or myoblast transfer in muscle in vivo.

Adenoviridae↗
PubMed1990

Molecular cloning and characterization of a late Tipula iridescent virus gene.

Virions of the cytoplasmic, icosahedral insect virus, Tipula iridescent virus (TIV), contain two major DNA components (L, greater than 176 kb; and S1, 10.8 kb) and 25-30 proteins. We characterized a gene (L96) whose 3.6-kb transcript is expressed late in the course of TIV infection of cultured of Estigmene acrea (salt marsh caterpillar, permissive host) and Aedes albopictus (mosquito, semipermissive host) cells. The L96 gene has an open reading frame of 867 codons, predicting a protein of 96 kDa with a pI of 10.9. The C terminus of the L96 protein is rich in hydrophobic amino acids and contains a small region of homology spanning a proteolytic cleavage site within two mammalian viral (GAG) polyproteins. Additional identity with H5 lysine-rich histones in the same region and with other DNA-binding proteins suggests that this protein may be involved in TIV structure. The lengths of the 5'- and 3'-untranslated regions of the L96 transcript were determined to be 21 nucleotides (nt) and 700 nt, respectively. Comparison of the TIV L96- and capsid-encoding genes, both of which are expressed late in infection, revealed that their 5' and 3' regions are generally rich in A and T residues, and that their 3' ends encode at least one eukaryotic polyadenylation signal (AATAAA).

Amino Acid Sequence↗
PubMed1990

Semipermissive replication of Tipula iridescent virus in Aedes albopictus C6/36 cells.

Comparative studies were carried out using two different insect cell lines, Aedes albopictus and Estigmene acrea, for Tipula iridescent virus (TIV) propagation. Light microscope autoradiography showed viral DNA present in viroplasmic centers (VCs) and an inhibition of nuclear DNA synthesis. These VCs appeared to be morphologically similar in both cell lines when examined by light and electron microscopy. Radiolabeled cDNA was synthesized from RNA samples obtained from infected cells at different times after infection and hybridized to TIV DNA digested with various restriction endonucleases. The results indicated that the pattern of transcription and the kinetics of TIV infection were qualitatively similar in both cell lines. The major TIV DNA components, L (greater than 174 kbp) and S1 (10.8 kbp) that are found in virions in approximately equivalent amounts, were made in both infected cell lines. However, the infected cell lines produced S1 DNA at higher levels relative to L than in virions. The cDNA hybridization studies also revealed that the S1 DNA has sequences that are transcribed and are TIV specific. While VC morphology, levels of L and S1 DNA synthesis, transcription, and capsid protein synthesis were similar in both cell lines, time course electron microscope studies revealed that progeny virions were detected only in the VCs of E. acrea cells and not in the VCs of A. albopictus cells, even by 96 hr p.i. These data suggest that the A. albopictus C6/36 cell line is semipermissive for TIV replication.

Aedes↗
PubMed1990

Molecular cloning, characterization, and expression of the Tipula iridescent virus capsid gene.

The capsid protein is the major structural component of the icosahedral Tipula iridescent virus (TIV) that replicates in cytoplasmic inclusion bodies of insect cells. TIV capsid protein purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was digested with trypsin and fractionated by reverse-phase high-pressure liquid chromatography. A mixed oligonucleotide constructed from the amino acid sequence of a capsid tryptic peptide was used for the identification and cloning of the corresponding gene. The single-copy capsid gene, located on a 2.47-kilobase-pair HindIII TIV genomic fragment, codes for a 464-amino-acid protein (50,831 daltons) with a predicted pI of 6.34. Analysis of total RNA from infected Estigmene acrea cells indicated that the 1.8-kilobase capsid transcript was maximally produced between 14 and 24 h after infection. Transcript mapping by primer extension indicated that the RNA start site was in the A+T-rich TGCTACTAAT sequence, 19 nucleotides upstream from the first ATG codon of the capsid open reading frame. Expression of the TIV capsid protein in infected E. acrea cells was demonstrated by in vivo labeling of total proteins with [35S]methionine, using anti-capsid antiserum as the probe. Capsid protein was also expressed in Escherichia coli cells by using a pUC19 plasmid containing a lacZ-capsid gene fusion.

Amino Acid Sequence↗
PubMedDate not supplied

Plaque assay and replication of Tipula iridescent virus in Spodoptera frugiperda ovarian cells.

A plaque assay was developed for the study of Tipula iridescent virus (TIV) replication using a cell line derived from the fall army worm Spodoptera frugiperda (Sf9). Infection and plaque formation were monitored with time by phase contrast microscopy, video and fluorescent light microscopy. Structure of virions, viroplasmic centres and organelles of infected cells were examined by transmission electron microscopy (TEM). After 4 h postinfection, plaques were visibly detected within the cell monolayer by the presence of localized cell damage and production of numerous vesicular-like cytoplasmic structures. Quantitation of virions present per A260 unit of TIV preparation was determined by TEM. The number of visible plaques corresponded to virus concentration and 1 A260 produced approximately 10(5) plaques. DNA hybridization analysis revealed no gross differences in genomic DNA from TIV propagated in either Sf9 cells or wax moth Galleria mellonella larvae. These findings indicate that Sf9 is permissive for replication of TIV and superior by some parameters to other cell lines currently in use for the study of host cell/TIV interactions.

Animals↗