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Expression of intermediate filament genes in myogenic lineage during mouse embryogenesis
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EXPRESSION OF INTERMEDIATE FILAMENT GENES IN MYOGENIC LINEAGE DURING MOUSE EMBRYOGENESIS
C o n t r o l o f m y o b l a s t c e l l adhe~dton a n d f u s i o n by a receptor-stllulated G-protein. R.E.Hausman & H. EIGendy, Boston University, Boston 9 M A 02215 U S A .
Zhen Lin LI, Hubert Condamine et Denise Paulin Universit~ Paris 7 et Institut Pasteur 25, rue du Dr Roux 75724 Paris Cedex 15 France
Differentiating embryonic chick m y o blasts undergo a characteristic increase in cell adhesion which is necessary for subsequent bilayer fusion and changes in membrane electrical properties. This increase in cell adhesion and subsequent steps in myoblast m e m b r a n e differentiation depend on prostaglandin (PG) binding to a transient m e m b r a n e receptor and require turnover of inositol phosphate. W e s h o w here that, at the time of P G binding, there is a pertussis toxin-sensitive event which is necessary for the increase in cell adhesion and further myogenic differentiation. Both before and after this time, myogenesis is not affected by the toxin. These observations are consistent with the P G receptor activating a G-protein which affects inoeitol polyphosphate metabolism. This G -protein is not sensitive to cholera toxin. W e are currently determining which other steps in myogenic gene expression are controlled by this P G initiated transductlon event.
Expression of intermediate filaments of the desmin end vimentin types has been studied in early mouse embryos. Desmin expression is first detected at day 9.5 pc in the myotomes. Thus, as was described in the case of cardiac actin, desrnln is expressed earlier than skeletal muscle genes, Whose expression does not start before day 10,5 pc in myotomes Structural and functional analysis of human desmin promoter shows the existence of a proximal element sufficient for expression, which contains sequences homologous to the cardiac actin promoter and of a distal element(s), which is characterized by the presence of CArG boxes, found in many mucle genes promoters, which is required for optimal expression in vitro (30 fold in transcriptional increase). The functional role of these elements in vivo for the developmental expression of desmln gene is being tested in vivo by mean of transgenic mice. In contrast to the in vitro situation where desmin expression is preceded by vlmentin expression, no vimentin can be detected in somhes and their derivatives including myotomes. Characterization of human vimentin promoter has been achieved in vitro and reveals the existence of multiple regulatory elements which can now be analysed in rive.
203
204
ONCOGENE MEDIATED INHIBITION OF MYOGENIC DIFFERENTIATION. A. Mendelsohn and J.R. Coleman, Brown University, Providence, RI, USA
THE DUCHENNE MUSCULAR DYSTROPHY GENE: STRUCTURE AND EXPRESSION IN MUSCLE AND NONMUSCLE TISSUES. David Yaffe, Dorit Zuk, Paz Einat, Adina Makover, Elisha Zeelon and Uri Nudel, Department of Cell Biology, The Weizmann Institute of Science, 76100 Rehovot, Israel
T h e basic objectives of this r e s e a r c h are to characterize the inhibition of differentiation that a c c o m p a n i e s oncogenic t r a n s f o r m a t i o n a n d to begin to delineate the m e c h a n i s m s of inhibition at the m o l e c u l a r level in cultured m a m m a l i a n myogenic cell lines. R a t L6 and m o u s e C2C12 myoblasts were t r a n s f e c t e d with a variety of o n c o g e n e constructs, using G418 resistance for selection of transfectants. Cells t r a n s f o r m e d by E l a , V-Fos, H a - R a s , a n d VM o s did not f o r m m y o t u b e s . Conversely, V-Myb, P53, V - E r b B, and V-Sis did not inhibit myogenesis. T h e r e s e e m s to be a correlation b e t w e e n transf o r m i n g activity of the o n c o g e n e clone in NIH 3T3 cells a n d the ability of an o n c o g e n e to s u p p r e s s differentiation. E x p r e s s i o n of MyoD1, a putative r e g u l a t o r of muscle cell differentiation, was dramatically lowered in E l a , H a - R a s , a n d V-Fos t r a n s f o r m e d C 2 C 1 2 cells, but not in p53 t r a n s f o r m e d C2C12 cells. This correlates with the ability of t h e s e lines to differentiate. L o w s e r u m significantly inhibited proliferation of v-Fos, H a - R a s , a n d E l a t r a n s f o r m e d cells, b u t did n o t stimulate differentiation, n o r did it re-establish n o r m a l levels of M y o D 1 expression. This suggests that s u p p r e s s i o n of differentiation by t h e s e o n c o g e n e s is probably not d u e to growth stimulation. (NSF-DCB-8609560)
The gene which, when defective, is responsible for Ouchenne muscular dystrophy (DMD) and Becket muscular dystrophy, is the largest gene known to date. It spans ca. 2000 kb of the x chromosome. The sequence of its 14 kb mRNA codes for 3685 amino acids. The predicted amino acid sequence, immunochemical and electron microscopy investigations suggest that the gene product is a structural protein associated with the cell membrane. The expression of the DMD gene is developmentally regulated in myogenic cells, similarly to the expression of muscle-specific genes such as myosin light chain 2 and skeletal muscle actin. However, significant amounts of the gene transcripts are also found in the brain and in several other nonmuscle tissues. RNAse protection assays and cDNA cloning revealed that the products of the gene in muscle and brain are not identical and that the 5' ends of the muscle and brain mRNAs are derived from 2 different exons, indicating that two or more promoters are involved in the regulation of transcription of the tissue-specific isoforms. The DNA regions containing the putative muscle and brain promoters have been isolated and sequenced. The muscle DMD promoter bears interesting similarities to the skeletal muscle actin promoter. A chimeric gene containing the muscle type DMD promoter and a reporter structural gene is developmentally regulated in transfected myogenic cells. Supported by the Muscular Dystrophy Association,the U.S.Israel BinationalScience Foundationand the Israel Academyof Science and the Humanities.
$65
202
EXPRESSION OF INTERMEDIATE FILAMENT GENES IN MYOGENIC LINEAGE DURING MOUSE EMBRYOGENESIS
C o n t r o l o f m y o b l a s t c e l l adhe~dton a n d f u s i o n by a receptor-stllulated G-protein. R.E.Hausman & H. EIGendy, Boston University, Boston 9 M A 02215 U S A .
Zhen Lin LI, Hubert Condamine et Denise Paulin Universit~ Paris 7 et Institut Pasteur 25, rue du Dr Roux 75724 Paris Cedex 15 France
Differentiating embryonic chick m y o blasts undergo a characteristic increase in cell adhesion which is necessary for subsequent bilayer fusion and changes in membrane electrical properties. This increase in cell adhesion and subsequent steps in myoblast m e m b r a n e differentiation depend on prostaglandin (PG) binding to a transient m e m b r a n e receptor and require turnover of inositol phosphate. W e s h o w here that, at the time of P G binding, there is a pertussis toxin-sensitive event which is necessary for the increase in cell adhesion and further myogenic differentiation. Both before and after this time, myogenesis is not affected by the toxin. These observations are consistent with the P G receptor activating a G-protein which affects inoeitol polyphosphate metabolism. This G -protein is not sensitive to cholera toxin. W e are currently determining which other steps in myogenic gene expression are controlled by this P G initiated transductlon event.
Expression of intermediate filaments of the desmin end vimentin types has been studied in early mouse embryos. Desmin expression is first detected at day 9.5 pc in the myotomes. Thus, as was described in the case of cardiac actin, desrnln is expressed earlier than skeletal muscle genes, Whose expression does not start before day 10,5 pc in myotomes Structural and functional analysis of human desmin promoter shows the existence of a proximal element sufficient for expression, which contains sequences homologous to the cardiac actin promoter and of a distal element(s), which is characterized by the presence of CArG boxes, found in many mucle genes promoters, which is required for optimal expression in vitro (30 fold in transcriptional increase). The functional role of these elements in vivo for the developmental expression of desmln gene is being tested in vivo by mean of transgenic mice. In contrast to the in vitro situation where desmin expression is preceded by vlmentin expression, no vimentin can be detected in somhes and their derivatives including myotomes. Characterization of human vimentin promoter has been achieved in vitro and reveals the existence of multiple regulatory elements which can now be analysed in rive.
203
204
ONCOGENE MEDIATED INHIBITION OF MYOGENIC DIFFERENTIATION. A. Mendelsohn and J.R. Coleman, Brown University, Providence, RI, USA
THE DUCHENNE MUSCULAR DYSTROPHY GENE: STRUCTURE AND EXPRESSION IN MUSCLE AND NONMUSCLE TISSUES. David Yaffe, Dorit Zuk, Paz Einat, Adina Makover, Elisha Zeelon and Uri Nudel, Department of Cell Biology, The Weizmann Institute of Science, 76100 Rehovot, Israel
T h e basic objectives of this r e s e a r c h are to characterize the inhibition of differentiation that a c c o m p a n i e s oncogenic t r a n s f o r m a t i o n a n d to begin to delineate the m e c h a n i s m s of inhibition at the m o l e c u l a r level in cultured m a m m a l i a n myogenic cell lines. R a t L6 and m o u s e C2C12 myoblasts were t r a n s f e c t e d with a variety of o n c o g e n e constructs, using G418 resistance for selection of transfectants. Cells t r a n s f o r m e d by E l a , V-Fos, H a - R a s , a n d VM o s did not f o r m m y o t u b e s . Conversely, V-Myb, P53, V - E r b B, and V-Sis did not inhibit myogenesis. T h e r e s e e m s to be a correlation b e t w e e n transf o r m i n g activity of the o n c o g e n e clone in NIH 3T3 cells a n d the ability of an o n c o g e n e to s u p p r e s s differentiation. E x p r e s s i o n of MyoD1, a putative r e g u l a t o r of muscle cell differentiation, was dramatically lowered in E l a , H a - R a s , a n d V-Fos t r a n s f o r m e d C 2 C 1 2 cells, but not in p53 t r a n s f o r m e d C2C12 cells. This correlates with the ability of t h e s e lines to differentiate. L o w s e r u m significantly inhibited proliferation of v-Fos, H a - R a s , a n d E l a t r a n s f o r m e d cells, b u t did n o t stimulate differentiation, n o r did it re-establish n o r m a l levels of M y o D 1 expression. This suggests that s u p p r e s s i o n of differentiation by t h e s e o n c o g e n e s is probably not d u e to growth stimulation. (NSF-DCB-8609560)
The gene which, when defective, is responsible for Ouchenne muscular dystrophy (DMD) and Becket muscular dystrophy, is the largest gene known to date. It spans ca. 2000 kb of the x chromosome. The sequence of its 14 kb mRNA codes for 3685 amino acids. The predicted amino acid sequence, immunochemical and electron microscopy investigations suggest that the gene product is a structural protein associated with the cell membrane. The expression of the DMD gene is developmentally regulated in myogenic cells, similarly to the expression of muscle-specific genes such as myosin light chain 2 and skeletal muscle actin. However, significant amounts of the gene transcripts are also found in the brain and in several other nonmuscle tissues. RNAse protection assays and cDNA cloning revealed that the products of the gene in muscle and brain are not identical and that the 5' ends of the muscle and brain mRNAs are derived from 2 different exons, indicating that two or more promoters are involved in the regulation of transcription of the tissue-specific isoforms. The DNA regions containing the putative muscle and brain promoters have been isolated and sequenced. The muscle DMD promoter bears interesting similarities to the skeletal muscle actin promoter. A chimeric gene containing the muscle type DMD promoter and a reporter structural gene is developmentally regulated in transfected myogenic cells. Supported by the Muscular Dystrophy Association,the U.S.Israel BinationalScience Foundationand the Israel Academyof Science and the Humanities.
$65