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Biomedical subjects

G Cossu

Publications and source records attributed to G Cossu.

At least 73 records · Page 4Linked to original sources
PubMed1998

Differential activation of Myf5 and MyoD by different Wnts in explants of mouse paraxial mesoderm and the later activation of myogenesis in the absence of Myf5.

Activation of myogenesis in newly formed somites is dependent upon signals derived from neighboring tissues, namely axial structures (neural tube and notochord) and dorsal ectoderm. In explants of paraxial mesoderm from mouse embryos, axial structures preferentially activate myogenesis through a Myf5-dependent pathway and dorsal ectoderm preferentially through a MyoD-dependent pathway. Here we report that cells expressing Wnt1 will preferentially activate Myf5 while cells expressing Wnt7a will preferentially activate MyoD. Wnt1 is expressed in the dorsal neural tube and Wnt7a in dorsal ectoderm in the early embryo, therefore both can potentially act in vivo to activate Myf5 and MyoD, respectively. Wnt4, Wnt5a and Wnt6 exert an intermediate effect activating both Myf5 and MyoD equivalently in paraxial mesoderm. Sonic Hedgehog synergises with both Wnt1 and Wnt7a in explants from E8.5 paraxial mesoderm but not in explants from E9.5 embryos. Signaling through different myogenic pathways may explain the rescue of muscle formation in Myf5 null embryos, which do not form an early myotome but later develop both epaxial and hypaxial musculature. Explants of unsegmented paraxial mesoderm contain myogenic precursors capable of expressing MyoD in response to signaling from a neural tube isolated from E10.5 embryos, the developmental stage when MyoD is present throughout the embryo. Myogenic cells cannot activate MyoD in response to signaling from a less mature neural tube. Together these data suggest that different Wnt molecules can activate myogenesis through different pathways such that commitment of myogenic precursors is precisely regulated in space and time to achieve the correct pattern of skeletal muscle development.

Animals↗
PubMed1998

Cellular adhesion in neoplastic and syngeneic normal cells under altered gravitational conditions.

The major objective of several experiments performed in space in the last 15 years was to establish whether single cells are sensitive to gravity. It was found in certain cells that reduced gravity leads to profound changes of a number of physiological functions like genetic expression, cell proliferation, signal transduction and cytoskeleton structure. In cell biology studies microgravity can be simulated on Earth in the clinostat. Nearly all data on experiments in the clinostat are related to cells cultured in suspension and, therefore, to adhesion-independent cells. In contrast, several biological phenomena as neoplastic transformation, cell differentiation, in-vitro cellular aging, contact inhibition and cellular adhesion require mainly cellular systems that are adhesion-dependent. The purpose of this work was: a) to study the behaviour of two rat cell strains (neoplastic SGS/4A and syngeneic fibroblasts FG) in order to test whether adhesion-dependent cells are suitable for clinorotation and b) to investigate cell-cell and cell-substratum adhesion in these cells kept under simulated low-g in the fast rotating clinostat and in hypergravity at l0g in the centrifuge.

Animals↗
PubMed1998

Microgravity simulations with human lymphocytes in the free fall machine and in the random positioning machine.

The purpose of this paper is to present the results obtained in our laboratory with both instruments, the FFM [free fall machine] and the RPM [random positioning machine], to compare them with the data from earlier experiments with human lymphocytes conducted in the FRC [fast rotating clinostat] and in space. Furthermore, the suitability of the FFM and RPM for research in gravitational cell biology is discussed.

Cells, Cultured↗
PubMed1997

Redefining the genetic hierarchies controlling skeletal myogenesis: Pax-3 and Myf-5 act upstream of MyoD.

We analyzed Pax-3 (splotch), Myf-5 (targeted with nlacZ), and splotch/Myf-5 homozygous mutant mice to investigate the roles that these genes play in programming skeletal myogenesis. In splotch and Myf-5 homozygous embryos, myogenic progenitor cell perturbations and early muscle defects are distinct. Remarkably, splotch/Myf-5 double homozygotes have a dramatic phenotype not seen in the individual mutants: body muscles are absent. MyoD does not rescue this double mutant phenotype since activation of this gene proves to be dependent on either Pax-3 or Myf-5. Therefore, Pax-3 and Myf-5 define two distinct myogenic pathways, and MyoD acts genetically downstream of these genes for myogenesis in the body. This genetic hierarchy does not appear to operate for head muscle formation.

Animals↗
PubMed1997

Rewarding properties of gamma-hydroxybutyric acid: an evaluation through place preference paradigm.

Gamma-hydroxybutyric acid (GHB), a putative neurotransmitter or neuromodulator found in the mammalian brain, has been successfully used in clinical practice to alleviate both alcohol and opiate withdrawal symptoms. In the present study we used a conditioned place preference (CPP) paradigm to investigate whether GHB possesses rewarding properties in rats. In order to exclude possible artifacts due to experimental design, we evaluated the possibility of a shift in preference when rats are conditioned either on their non-preferred side or on a randomly assigned side of conditioning. In both experiments GHB was seen to induce CPP. Although to date the physiological role of this compound still remains unclear, there is no doubt that GHB, further to its proven effect on alcohol and opiates, possesses rewarding properties of its own. The abuse liability afforded by this drug suggests the use of particular caution in handling GHB as a clinically useful drug.

Animals↗
PubMed1997

Establishing myogenic identity during somitogenesis.

Over the past year, interest has focused on identifying signalling molecules--including Wnts, Sonic hedgehog, BMP-4, and noggin--that divert somitic mesodermal cells into the muscle lineage, either by induction or derepression. New mouse mutants have also provided insights into somite formation and differentiation, as well as pointing to novel differences between head, trunk, and limb myogenic programmes. In addition, recent genetic, embryological, and molecular studies have shed new light on somite formation and the establishment of muscle progenitor cells.

Animals↗
PubMed1997

Localization of beta-D-galactosidase activity in semithin epon sections of embryonic tissues using differential interference contrast optics.

In the present study we describe a method for the histochemical demonstration of beta-D-galactosidase activity on tissue sections processed for light microscopy at high resolution. 5-Bromo-indolyl-beta-D-galactopyranoside (Bluo-Gal) was utilized as an indigogenic method for the demonstration of Escherichia coli beta-D-galactosidase reporter gene activity whose expression was studied in a transgenic line where the enzyme, with a nuclear localization signal (nlacZ), is under the transcriptional control of a striated muscle-specific promoter. At the light-microscopic level, by using Differential Interference Contrast (DIC) optics, the reaction product was detected as precipitates in the form of fine birefringent crystals. These were located around and inside the nuclei of beta-gal-expressing cells. This simple method allows an easy and rapid identification of few or even one labeled cell(s) within large microscopic fields (whole embryos) and the labeled cell(s) can be evaluated both morphologically and quantitatively.

Animals↗
PubMed1997

Absence of MEF2 binding to the A/T-rich element in the muscle creatine kinase (MCK) enhancer correlates with lack of early expression of the MCK gene in embryonic mammalian muscle.

During skeletal muscle development, different types of muscle fibers are generated, which express different combinations of muscle-specific gene products. For example, the muscle creatine kinase gene (MCK) is highly expressed in fetal but not embryonic myotubes. We performed transient transfections of CAT reporter constructs, driven by the MCK promoter with variable lengths of 5'-flanking sequence, into primary cultures of embryonic and fetal muscle cells. Reporter activity was observed in fetal but not embryonic muscle cells. We assayed the ability of nuclear extracts prepared from embryonic and fetal muscle and C2C12 myotubes to bind specific regulatory elements in the MCK enhancer. The profile of DNA/protein complexes resulting from electrophoretic mobility shift assays was qualitatively the same with all extracts used when the oligonucleotide probes represented the MCK-E-box, MHox site, CArG-box, and AP2 site. In contrast, no binding activity to the MEF2 site was observed with embryonic nuclear extract. Interestingly, MEF2 mRNAs and proteins were detected in both fetal and embryonic muscle, with the exception of the MEF2D1b isoform, which is restricted to fetal muscle. Furthermore, we found that protein phosphatase inhibitors included in the preparation of embryonic nuclear extracts or added to the medium of transfected embryonic myotubes can restore MEF2 DNA binding activity, as well as reporter activity driven by the MCK promoter and partial transcriptional activation of the endogenous MCK gene. We propose that phosphorylation of MEF2 regulates its activity and represents an important aspect of the mechanism controlling stage-specific transcription during skeletal myogenesis.

Alkaline Phosphatase↗
PubMed1997

A temperature-conditional mutant of simian virus 40 large T antigen requires serum to inhibit myogenesis and does not induce DNA synthesis in myotubes.

The temperature-conditional mutant tsA58 of SV40 large T antigen (Tag) increases the proliferation rate and the number of cell divisions in primary murine and human myogenic cells when expressed under permissive conditions (i.e., at 33 degrees C in medium containing high levels of serum). Under these conditions, Tag also prevents terminal differentiation. Under nonpermissive conditions (i.e., at 39 degrees C in medium containing low levels of serum) in which Tag is largely inactive, proliferation is arrested, and differentiation occurs. However, even at a permissive temperature, the removal of serum induced myosin expression and the fusion of myogenic cells, which continued to express functional Tag. Although Tag was complexed with pRb, as expected from a functional protein, proliferation was nevertheless arrested, and differentiation was induced. Consistent with these findings, the exposure of Tag-expressing differentiated myotubes to serum at 33 degrees C did not reinduce DNA synthesis in these cells. Thus, in myogenic cells, temperature-conditional mutants of Tag stimulate proliferation in the presence of serum but neither prevent terminal differentiation in the absence of serum nor induce DNA synthesis once complete withdrawal from the cycle has occurred.

Animals↗
PubMed1997

Unorthodox myogenesis: possible developmental significance and implications for tissue histogenesis and regeneration.

During the last few years several reports have described the occurrence of skeletal myogenesis in cells derived from embryonic, fetal and perinatal tissues that usually do not contribute to skeletal muscle in the adult vertebrate body. After a brief description of current ideas on myogenic determination in higher vertebrates, three examples of this unorthodox myogenesis will be described: 1) the occurrence of myogenesis in chick epiblast cells, cultured in isolation in serum-free medium; 2) the presence of cells endowed with myogenic potential in the embryonic mouse neural tube; and 3) the occurrence of spontaneous or induced myogenesis in mesenchymal cells during fetal and postnatal life. A possible embryological basis for unorthodox myogenesis, in relation to gastrulation and morphogenetic fields, is then presented. It is also proposed that unorthodox myogenesis may represent a compensatory mechanism for higher vertebrates that have lost much of the regeneration potential of lower vertebrates.

Animals↗
PubMed1996

The inhibition of differentiation caused by TGFbeta in fetal myoblasts is dependent upon selective expression of PKCtheta: a possible molecular basis for myoblast diversification during limb histogenesis.

Embryonic and fetal skeletal myoblasts are responsible for the formation of primary and secondary fibers in mammals, but the mechanism which diversifies their fate is unknown. In vitro, embryonic myoblasts are resistant to the differentiation inhibitory effects of transforming growth factor beta and phorbol esters. Thus, differential expression of specific molecules involved in the transduction of extracellular signals may contribute to the different phenotypes. We report here that protein kinase C theta, but none of the other known protein kinase C isoforms, is selectively expressed in fetal and postnatal muscle cells (at both the myoblast and myotube stage) in vitro and in vivo. By contrast, embryonic myoblasts and myotubes do not express protein kinase C theta in vitro or in vivo. This difference is causally related to a differential response to transforming growth factor beta, since overexpression of protein kinase C theta, but not of protein kinase C alpha or zeta, in embryonic myoblasts makes these cells sensitive to transforming growth factor beta. These data demonstrate for the first time that a protein kinase C isoform is a key component of the signal transduction cascade which follows exposure of myoblasts to transforming growth factor beta. They also suggest a specific role for protein kinase C theta in determining the fate of different myoblasts during muscle histogenesis.

Amino Acid Sequence↗
PubMed1996

Rh D/d genotyping by quantitative polymerase chain reaction and capillary zone electrophoresis.

A safe and reliable method for determining RhD type (positive or negative) and zygosity (D/D or D/d) could have applications, for instance, in the prediction of the D genotype of a father in couples where there is an RhD-negative woman at risk of fetal alloimmunization. Capillary zone electrophoresis (CZE) is proposed as a novel, reliable and powerful method for quantitative evaluation of polymerase chain reaction (PCR) products. RhD is determined by amplifying a 136 bp region common to the RhCcEe and RhD genes and a 186 bp region specific of the RhD gene. RhD positive and negative samples are identified by polyacrylamide gel slab electrophoresis, followed by silver staining of the DNA bands, and by CZE in sieving liquid polymers, with direct on-line peak densitometric evaluation by exploiting the intrinsic UV absorbance of the DNA fragments at 254 nm. The CZE method allows not only a reliable assessment of the RhD type (the presence of both 136 bp and 186 bp fragments indicating an RhD-positive type, the presence of only the 136 bp fragment indicating an RhD-negative type), but also a rapid determination of the zygosity based on the quantitative expression ratio of the 136 bp/186 bp pair. Thus, a 2:1 peak ratio clearly indicates a D/D homozygous individual, whereas a 3:1 peak ratio gives evidence of a D/d heterozygous individual.

DNA↗
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

Differentiation dependent expression in muscle cells of ZT3, a novel zinc finger factor differentially expressed in embryonic and adult tissues.

ZT3, isolated from a murine muscle cell cDNA library by a low-stringency hybridization, encodes a zinc finger domain containing factor with a transcript of 5.0 kb. A 3' 2.5 kb partial nucleotide sequence contains an ORF of 1.5 kb where 17 canonical C2H2 zinc finger domains organized in tandem were identified. It maps on mouse chromosome 11, close to two mutations which affect skeletal formation. ZT3 expression depends upon differentiation of myogenic cells in culture, since it is upregulated with myogenin and inhibited in scr-transfected C2C12 cells. ZT3 is not expressed in NIH3T3 or C3H10T1/2 fibroblasts, but is induced when fibroblasts are myogenically converted by transfection with the muscle regulatory genes (MRFs). Its expression is also upregulated in the rhabdomyosarcoma cell line RD induced to myogenic differentiation by TPA treatment. In postimplantation embryos, ZT3 is diffusely expressed but higher expression is detectable in the neural tube and encephalic vesicles, in the somites and, at a high level, in the limb buds as they form. During further development ZT3 is expressed in many tissues of neuroectodermal and mesodermal origin, but its expression decreases during fetal development and in the adult it is restricted to skeletal and cardiac muscle and to spleen. This pattern of expression suggests a possible role played by ZT3 in differentiating skeletal muscle. Its expression in other tissues is compatible with the suggestion that members of this class of DNA-binding factors play different roles during post-implantation development and in the adult life.

3T3 Cells↗
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

Gene dosage in capillary electrophoresis: pre-natal diagnosis of Down's syndrome.

Modern proposals for pre-natal genetic analysis of Down's syndrome consist in isolating DNA from amniotic cells and amplifying a highly polymorphic small tandem repeat region of the chromosome 21-specific D21S11 marker. The polymerase-chain-reaction-amplified fragments are typically 5'-end labelled with a green or blue fluorescent reporter and data acquisition occurs on-lane in DNA sequencing gel-slabs and equipment. The following patterns are expected: for normal individuals, 1 peak or two peaks in a 1:1 ratio. In the case of trisomy 21, the following patterns are found: either three peaks in a 1:1:1 ratio or a two-peak profile with a 2:1 gene ratio. We have developed a capillary electrophoretic system, offering precise diagnostic value by exploiting the intrinsic DNA absorbance at 254 nm. The separation occurs in capillaries coated with an extremely stable and hydrophilic layer of poly(N-acroyloyl amino ethoxy ethanol) and filled with a background electrolyte consisting of 89 mM Tris-borate, 2 mM EDTA, 2.5 microM ethidium bromide and 8% short-chain, low-viscosity, replaceable, liquid, linear, sieving polyacrylamide. The technique offers high reproducibility and precise on-line, automated peak acquisition and quantitation.

DNA Primers↗
PubMed1995

Myoblast differentiation during mammalian somitogenesis is dependent upon a community effect.

The differentiation potential of early mammalian myogenic cells was tested under clonal culture conditions. Cells were isolated from paraxial mesoderm and limb buds of transgenic mouse embryos at 9.5 days after conception and grown in culture at clonal density either on collagen-coated dishes or on various feeder cell layers. The transgene used contained a reporter gene encoding beta-galactosidase with a nuclear localization signal under the control of regulatory sequences from the gene for fast myosin light chain 3, so that beta-galactosidase staining indicated the presence of differentiated muscle cells. After 5 days in culture, the number and size of beta-galactosidase-positive (beta-gal+) clones were recorded. Cells isolated from somites I-V (the last five somites to have formed) or from unsegmented paraxial mesoderm did not give rise to any beta-gal+ clones. Cells isolated from somites VI-X or from the forelimb bud gave rise to beta-gal+ clones, but only on feeder cells. Cells from somites XI or older gave rise to beta-gal+ clones independently of the substrate. However, when cells isolated from unsegmented paraxial mesoderm or somites I-V were cultured with nontransgenic cells from the trunk (including neural tube and notochord), differentiation occurred on condition that the cells were in a three-dimensional aggregate, even though their specific position in the somite had been lost. By culturing explants ranging in size from 1 to < 100 cells in the presence of an inhibitor of cell division, we determined that a minimal number of 30-40 cells is required for mesodermal cells to differentiate.

Animals↗