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.

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.

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.

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.

MyoD1: a nuclear phosphoprotein requiring a Myc homology region to convert fibroblasts to myoblasts.

Expression of a complementary DNA (cDNA) encoding the mouse MyoD1 protein in a variety of fibroblast and adipoblast cell lines converts them to myogenic cells. Polyclonal antisera to fusion proteins containing the MyoD1 sequence show that MyoD1 is a phosphoprotein present in the nuclei of proliferating myoblasts and differentiated myotubes but not expressed in 10T1/2 fibroblasts or other nonmuscle cell types. Functional domains of the MyoD1 protein were analyzed by site-directed deletional mutagenesis of the MyoD1 cDNA.

Differential binding of antibodies against the neurofilament triplet proteins in different avian neurons.

Using monospecific antisera against each of the three chicken neurofilament (NF) proteins, NF70, NF160 and NF180, the distribution of each of these proteins in several types of neurons was examined by immunohistochemistry. Striking differences were observed in the relative staining by the three antibodies when the soma of different types of neurons were compared, and also when the soma of some neurons were compared with their axons. Both the soma and axons of dorsal root sensory neurons were brightly stained by each of the antisera.

Effects of phorbol 12-myristate 13-acetate on the differentiation program of embryonic chick skeletal myoblasts.

The effects of phorbol 12-myristate 13-acetate (PMA) on three aspects of myogenesis have been analyzed: (a) fusion of mononucleated myogenic cells to form myotubes; (b) synthesis and accumulation of two muscle-specific proteins; and (c) DNA synthesis. Using autoradiography combined with immunofluorescent localization of muscle-specific light meromyosin and the muscle-specific intermediate filament protein desmin, we have found that embryonic chick myogenic cells cultured in the presence of PMA (50 nM) initiate the synthesis of both desmin and muscle-specific light meromyosin and, by these criteria, partially differentiate. These cells differ from normal definitive postmitotic myoblasts, however, since they (a) do not fuse; (b) do not assemble normal myofibrils; and (c) incorporate [3H]thymidine.

Taxol induces postmitotic myoblasts to assemble interdigitating microtubule-myosin arrays that exclude actin filaments.

Taxol has the following effects on myogenic cultures: (a) it blocks cell replication of presumptive myoblasts and fibroblasts. (b) It induces the aggregation of microtubules into sheets or massive cables in presumptive myoblasts and fibroblasts, but not in postmitotic, mononucleated myoblasts. (c) It induces normally elongated postmitotic myoblasts to form stubby, star-shaped cells.

Different proteins associated with 10-nanometer filaments in cultured chick neurons and nonneuronal cells.

A protein of molecular size 180 kilodaltons is associated with 10-nanometer filaments in neurons and is immunologically distinct from smaller putative neurofilament subunits and from 10-nanometer filament proteins in nonneuronal cells, such as myotubes and fibroblasts. Neurons do not contain vimentin, the major filament protein in many other cells, including the nonneuronal cells in cultures of neural tissue.

Differential location of different types of intermediate-sized filaments in various tissues of the chicken embryo.

The location of constitutive proteins of different types of intermediate-sized (about 10 mm) filaments (cytokeratin, vimentin, desmin, brain filament protein) was examined in various tissues of 11--20 day chick embryos, using specific antibodies against the isolated proteins and immunofluorescence microscopy on frozen sections and on isolated serous membrane. The tissues studied which contained epithelia were small intestine, gizzard, esophagus, crop, liver, kidney, thymus, mesenteries, and epidermis. The results show that the different intermediate filament proteins, as seen in the same organ, are characteristic of specific lines of differentiation: Cytokeratin filaments are restricted to--and specific for--epithelial cells; vimentin filaments are seen--at this stage of embryogenesis--only in mesenchymal cells, including connective tissue, endothelial and blood cells, and chondrocytes; filaments containing protein(s) related to the subunit protein prepared from gizzard 10 nm filaments (i.