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A B Lassar

Publications and source records attributed to A B Lassar.

At least 37 records · Page 2Linked to original sources
PubMed1995

Combinatorial signaling by Sonic hedgehog and Wnt family members induces myogenic bHLH gene expression in the somite.

We have demonstrated previously that a combination of signals from the neural tube and the floor plate/notochord complex synergistically induce the expression of myogenic bHLH genes and myogenic differentiation markers in unspecified somites. In this study we demonstrate that Sonic hedgehog (Shh), which is expressed in the floor plate/notochord, and a subset of Wnt family members (Wnt-1, Wnt-3, and Wnt-4), which are expressed in dorsal regions of the neural tube, mimic the muscle inducing activity of these tissues. In combination, Shh and either Wnt-1 or Wnt-3 are sufficient to induce myogenesis in somitic tissue in vitro. Therefore, we propose that myotome formation in vivo may be directed by the combinatorial activity of Shh secreted by ventral midline tissues (floor plate and notochord) and Wnt ligands secreted by the dorsal neural tube.

Animals↗
PubMed1995

Correlation of terminal cell cycle arrest of skeletal muscle with induction of p21 by MyoD.

Skeletal muscle differentiation entails the coordination of muscle-specific gene expression and terminal withdrawal from the cell cycle. This cell cycle arrest in the G0 phase requires the retinoblastoma tumor suppressor protein (Rb). The function of Rb is negatively regulated by cyclin-dependent kinases (Cdks), which are controlled by Cdk inhibitors. Expression of MyoD, a skeletal muscle-specific transcriptional regulator, activated the expression of the Cdk inhibitor p21 during differentiation of murine myocytes and in nonmyogenic cells. MyoD-mediated induction of p21 did not require the tumor suppressor protein p53 and correlated with cell cycle withdrawal. Thus, MyoD may induce terminal cell cycle arrest during skeletal muscle differentiation by increasing the expression of p21.

Animals↗
PubMed1995

Inhibition of myogenic differentiation in proliferating myoblasts by cyclin D1-dependent kinase.

Although the myogenic regulator MyoD is expressed in proliferating myoblasts, differentiation of these cells is limited to the G0 phase of the cell cycle. Forced expression of cyclin D1, but not cyclins A, B, or E, inhibited the ability of MyoD to transactivate muscle-specific genes and correlated with phosphorylation of MyoD. Transfection of myoblasts with cyclin-dependent kinase (Cdk) inhibitors p21 and p16 augmented muscle-specific gene expression in cells maintained in high concentrations of serum, suggesting that an active cyclin-Cdk complex suppresses MyoD function in proliferating cells.

Animals↗
PubMed1995

Induction of avian cardiac myogenesis by anterior endoderm.

An experimental system was devised to study the mechanisms by which cells become committed to the cardiac myocyte lineage during avian development. Chick tissues from outside the fate map of the heart (in the posterior primitive streak (PPS) of a Hamburger & Hamilton stage 4 embryo) were combined with potential inducing tissues from quail embryos and cultured in vitro. Species-specific RT-PCR was employed to detect the appearance of the cardiac muscle markers chick Nkx-2.5 (cNkx-2.5), cardiac troponin C and ventricular myosin heavy chain in the chick responder tissues. Using this procedure, we found that stage 4-5 anterior lateral (AL) endoderm and anterior central (AC) mesendoderm, but not AL mesoderm or posterior lateral mesendoderm, induced cells of the PPS to differentiate as cardiac myocytes. Induction of cardiogenesis was accompanied by a marked decrease in the expression of rho-globin, implying that PPS cells were being induced by anterior endoderm to become cardiac myocytes instead of blood-forming tissue. These results suggest that anterior endoderm contains signaling molecules that can induce cardiac myocyte specification of early primitive streak cells. One of the cardiac muscle markers induced by anterior endoderm, cNkx-2.5, is here described for the first time. cNkx-2.5 is a chick homeobox-containing gene that shares extensive sequence similarity with the Drosophila gene tinman, which is required for Drosophila heart formation. The mesodermal component of cNkx-2.5 expression from stage 5 onward, as determined by in situ hybridization, is strikingly in accord with the fate map of the avian heart. By the time the myocardium and endocardium form distinct layers, cNkx-2.5 is found only in the myocardium. cNkx-2.5 thus appears to be the earliest described marker of avian mesoderm fated to give rise to cardiac muscle.

Amino Acid Sequence↗
PubMed1995

Combinatorial signals from the neural tube, floor plate and notochord induce myogenic bHLH gene expression in the somite.

The neural tube, floor plate and notochord are axial tissues in the vertebrate embryo which have been demonstrated to play a role in somite morphogenesis. Using in vitro coculture of tissue explants, we have monitored inductive interactions of these axial tissues with the adjacent somitic mesoderm in chick embryos. We have found that signals from the neural tube and floor plate/notochord are necessary for expression of the myogenic bHLH regulators MyoD, Myf5 and myogenin in the somite. Eventually somitic expression of the myogenic bHLH genes is maintained in the absence of the axial tissues. In organ culture, at early developmental stages (HH 11-), induction of myogenesis in the three most recently formed somites can be mediated by the neural tube together with the floor plate/notochord, while in more rostral somites (stages IV-IX) the neural tube without the floor plate/notochord is sufficient. By recombining somites and neural tubes from different axial levels of the embryo, we have found that a second signal is necessary to promote competence of the somite to respond to inducing signals from the neural tube. Thus, we propose that at least two signals from axial tissues work in combination to induce myogenic bHLH gene expression; one signal derives from the floor plate/notochord and the other signal derives from regions of the neural tube other than the floor plate.

Animals↗
PubMed1992

A novel myoblast enhancer element mediates MyoD transcription.

The MyoD gene can orchestrate the expression of the skeletal muscle differentiation program. We have identified the regions of the gene necessary to reproduce transcription specific to skeletal myoblasts and myotubes. A proximal regulatory region (PRR) contains a conserved TATA box, a CCAAT box, and a GC-rich region that includes a consensus SP1 binding site. The PRR is sufficient for high levels of skeletal muscle-specific activity in avian muscle cells. In murine cells the PRR alone has only low levels of activity and requires an additional distal regulatory region to achieve high levels of muscle-specific activity. The distal regulatory region differs from a conventional enhancer in that chromosomal integration appears necessary for productive interactions with the PRR. While the Moloney leukemia virus long terminal repeat can enhance transcription from the MyoD PRR in both transient and stable assays, the simian virus 40 enhancer cannot, suggesting that specific enhancer-promoter interactions are necessary for PRR function.

Animals↗
PubMed1991

Functional activity of myogenic HLH proteins requires hetero-oligomerization with E12/E47-like proteins in vivo.

In this report we provide four lines of evidence indicating that E12/E47-like proteins interact in vivo with the myogenic HLH proteins MyoD and myogenin. First, cotransfection of MyoD and E47 in COS cells indicates that these factors synergistically enhance transcription of a reporter gene containing an oligomerized MyoD-binding site. Second, mobility-shift assays of muscle cell nuclear extracts, "double shifted" with specific antisera, have identified complexes binding to the MEF1 site that contain either MyoD or myogenin in association with E12/E47-like proteins. Third, association with E47 alters the phosphorylation state of MyoD. Fourth, C3H10T1/2 cells expressing antisense E2A transcripts contain low levels of E2A gene products and display less terminal muscle differentiation when infected with retroviral MyoD or when challenged to differentiate with 5-azacytidine treatment. In addition we demonstrate that MyoD, in conjunction with E12/E47-like proteins, is functioning as a regulatory nodal point for activation of several other downstream muscle regulators.

Adenovirus Early Proteins↗
PubMed1990

The MyoD DNA binding domain contains a recognition code for muscle-specific gene activation.

A 60 amino acid domain of the myogenic determination gene MyoD is necessary and sufficient for sequence-specific DNA binding in vitro and myogenic conversion of transfected C3H10T1/2 cells. We show that a highly basic region, immediately upstream of the helix-loop-helix (HLH) oligomerization motif, is required for MyoD DNA binding in vitro. Replacing helix1, helix2, or the loop of MyoD with the analogous sequence of the Drosophila T4 achaete-scute protein (required for peripheral neurogenesis) has no substantial effect on DNA binding in vitro or muscle-specific gene activation in transfected C3H10T1/2 cells. However, replacing the basic region of MyoD with the analogous sequence of other HLH proteins (the immunoglobulin enhancer binding E12 protein or T4 achaete scute protein) allows DNA binding in vitro, yet abolishes muscle-specific gene activation. These findings suggest that a recognition code that determines muscle-specific gene activation lies within the MyoD basic region and that the capacity for specific DNA binding is insufficient to activate the muscle program.

Amino Acid Sequence↗
PubMed1990

MyoD induces growth arrest independent of differentiation in normal and transformed cells.

MyoD is a gene involved in the control of muscle differentiation. We show that MyoD causes growth arrest when expressed in cell lines derived from tumors or transformed by different oncogenes. MyoD-induced growth inhibition was demonstrated by reduction in the efficiency of colony formation and at the single-cell level. We further show that MyoD growth inhibition can occur in cells that are not induced to activate muscle differentiation markers. The inhibitory activity of MyoD was mapped to the same 68-amino acid segment necessary and sufficient for induction of muscle differentiation, the basic-helix-loop-helix motif. Mutants with alterations in the basic region of MyoD that fail to bind or do not activate a muscle-specific enhancer inhibited growth; mutants with deletions in the helix-loop-helix region failed to inhibit growth. Thus, inhibition of cell growth by MyoD seems to occur by means of a parallel pathway to the one that leads to myogenesis. We conclude that MyoD is a prototypic gene capable of functionally activating intracellular growth inhibitory pathways.

Animals↗
PubMed1989

MyoD is a sequence-specific DNA binding protein requiring a region of myc homology to bind to the muscle creatine kinase enhancer.

MyoD is a skeletal muscle-specific protein that is able to induce myogenesis in a wide variety of cell types. In this report, we show that MyoD is a DNA binding protein capable of specific interaction with two regions of the mouse muscle creatine kinase gene upstream enhancer, both of which are required for full muscle-specific enhancer activity. MyoD shares antigenicity and DNA binding specificity with MEF1, a myocyte-specific DNA binding factor. The contiguous basic and myc homology regions of MyoD that are necessary and sufficient for specific DNA interaction are the same regions of the protein required to convert 10T1/2 fibroblasts into muscle. These findings suggest that the biological activity of MyoD is mediated via its capacity for specific DNA interaction.

Animals↗
PubMed1989

Transformation by activated ras or fos prevents myogenesis by inhibiting expression of MyoD1.

Transformation of myoblasts by activated ras inhibits myogenic differentiation. We demonstrate that this oncogene inhibits expression of the muscle regulatory factors MyoD1 and myogenin. Expression of retroviral-encoded MyoD1 in ras-transformed myoblasts leads to the re-expression of both terminal differentiation markers and lineage markers expressed in proliferating myoblasts (including endogenous MyoD1 and myogenin), suggesting that ras inhibits myogenic differentiation in a manner dependent on the loss of MyoD1 expression. In addition, we show that fos transformation of myoblasts inhibits muscle differentiation by a similar mechanism.

Animals↗
PubMed1989

Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence.

A DNA binding and dimerization motif, with apparent amphipathic helices (the HLH motif), has recently been identified in various proteins, including two that bind to immunoglobulin enhancers (E12 and E47). We show here that various HLH proteins can bind as apparent heterodimers to a single DNA motif and also, albeit usually more weakly, as apparent homodimers. The HLH domain can mediate heterodimer formation between either daughterless, E12, or E47 (Class A) and achaete-scute T3 or MyoD (Class B) to form proteins with high affinity for the kappa E2 site in the immunoglobulin kappa chain enhancer. The achaete-scute T3 and MyoD proteins do not form kappa E2-binding heterodimers together, and no active complex with N-myc was evident. The formation of a heterodimer between the daughterless and achaete-scute T3 products may explain the similar phenotypes of mutants at these two loci and the genetic interactions between them. A role of E12 and E47 in mammalian development, analogous to that of daughterless in Drosophila, is likely.

Animals↗
PubMed1989

5-bromo-2'-deoxyuridine blocks myogenesis by extinguishing expression of MyoD1.

The pyrimidine analog 5-bromodeoxyuridine (BUdR) competes with thymidine for incorporation into DNA. Substitution of BUdR for thymidine does not significantly affect cell viability but does block cell differentiation in many different lineages. BUdR substitution in a mouse myoblast line blocked myogenic differentiation and extinguished the expression of the myogenic determination gene MyoD1. Forced expression of MyoD1 from a transfected expression vector in a BUdR-substituted myoblast overcame the block to differentiation imposed by BUdR. Activation of BUdR-substituted muscle structural genes and apparently normal differentiation were observed in transfected myoblasts. This shows that BUdR blocks myogenesis at the level of a myogenic regulatory gene, possibly MyoD1, not by directly inhibiting the activation of muscle structural genes. It is consistent with the idea that BUdR selectively blocks a class of regulatory genes, each member of which is important for the development of a different cell lineage.

Animals↗
PubMed1989

Positive autoregulation of the myogenic determination gene MyoD1.

Transfection of cDNA expression vectors encoding either MyoD1 or myogenin into 10T1/2 cells converts them to myogenic cells. We show that transfection of 10T1/2 cells with the MyoD1 cDNA activates expression of endogenous MyoD1 mRNA, indicating that MyoD1 is subject to positive autoregulation. This activation of endogenous MyoD1 mRNA was also observed in Swiss 3T6 cells, but not in several other fibroblast or adipoblast cell lines transfected with the MyoD1 cDNA. In addition, transfection of the MyoD1 cDNA leads to activation of myogenin expression, and transfection of the myogenin cDNA leads to activation of MyoD1 expression. Thus, MyoD1 and myogenin appear to function in a positive autoregulatory loop that could either: account for or contribute to the stability of myogenic commitment; or amplify the level of expression of both MyoD1 and myogenin above a critical threshold that is required for activation of the myogenic program.

Animals↗
PubMed1989

Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD.

MyoD is a master regulatory gene for myogenesis. Under the control of a retroviral long terminal repeat, MyoD was expressed in a variety of differentiated cell types by using either a DNA transfection vector or a retrovirus. Expression of muscle-specific proteins was observed in chicken, human, and rat primary fibroblasts and in differentiated melanoma, neuroblastoma, liver, and adipocyte lines. The ability of MyoD to activate muscle genes in a variety of differentiated cell lines suggests that no additional tissue-specific factors other than MyoD are needed to activate the downstream program for terminal muscle differentiation or that, if such factors exist, they are themselves activated by MyoD expression.

Adipose Tissue↗
PubMed1989

MyoD and achaete-scute: 4-5 amino acids distinguishes myogenesis from neurogenesis.

The myogenic determination gene MyoD contains a 60 amino acid domain that is necessary for both sequence-specific DNA binding and myogenic conversion of transfected C3H10T1/2 mouse embryo fibroblasts. We have generated deletion, insertion, and substitution mutants to probe the structure and function of this region, both in vitro and in vivo. Our results are consistent with a previous proposal that a helix-loop-helix (HLH) motif mediates protein dimerization. A highly basic region located immediately upstream of the HLH motif is required for DNA binding, which occurs if and only if the HLH motif is capable of dimerization. All mutants of MyoD that do not bind DNA in vitro fail to activate myogenesis or expression of a co-transfected muscle-specific reporter gene in transfected C3H10T1/2 cells. Replacing either helix 1, helix 2, or the loop sequence of MyoD with the analogous sequence of the Drosophila T4 achaete-scute protein (which is required for neurogenesis) has no substantial effect on DNA binding or biological activity. However, replacing the basic region of MyoD with the analogous sequence of the kappa immunoglobulin enhancer binding protein E12, or the T4 achaete-scute protein, produces proteins still capable of specific DNA binding in vitro, yet without significant biological activity. These findings suggest that within the 13 amino acid sequence of the MyoD basic region lies a recognition code that determines muscle-specific gene expression, although specific DNA binding per se is not sufficient to activate the muscle program.

Amino Acid Sequence↗