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The cellular basis for commitment in skeletal muscle and for diversity of myosin heavy chain expression in avian development
J Mol Cell Cardiol 19 (Supplement IV) (1987)
49
AUTONOMOUS DIFFERENTIATION OF CARDIAC MYOBLASTS IN VITRO. D. Bader, T. Schultheiss, H. Holtzer, F. Wachtler. Dept. of Cell Biology and Anatomy, Cornell University Medical College, NY, NY. Dept. of Anatomy, University of Pennsylvannia, Philadelphia, PA. The central portion of blastoderms of the chicken embryo was dissociated into single cells and cultured at high or low density for periods up to five weeks. Clusters of rhythmically beating cells were first observed on the forth day of culture. Cultures were fixed and screened for the presence of sarcomeric myosin heavy chains with a monoclonal antibody which recognizes all sarcomeric myosins and monoclona) antibodies specific for atria), ventricular or conduction system isoforms. In cultures plated both at an initially high or low density cells expressing sarcomeric myosin could be found either as single cells or as clusters. Only in those cultures plated at an initially high density were atrial and ventricular specific isoforms detected, whereas cardiac myocytes in low density cultures as well did not express myosin heavy chains reactive with our tissue specific antibodies. No cells containing conduction system specific myosin isoforms were ever seen in any of our cultures. Addition of TPA during the initial period of culture ( 41 day) prevented the emergence of cardiac myocytes as determined by the absence of beating ceils and sarcomeric myosin heavy chain positive cells. Adding TPA after the onset of cardiac myogenesis did not alter the expression of myosin heavy chains, We conclude that cardiac myoblasts differentiate and diversify in the absence of heart morphogenesis.
50
THE CELLULAR BASIS FOR COMMITMENT IN SKELETAL MUSCLE AND FOR DIVERSITY OF MYOSIN HEAVY CHAIN EXPRESSION IN AVIAN DEVELOPMENT. F.E. Stockdale, ~].B. Miller. Department of Medicine/Oncology Stanford University Medical School, Stanford, California. Diversification of cell types in the embryo is attributed to differences in commitment among the precursor cells that produce different cell types. This concept has not been used to explain the origins of different skeletal muscle fiber types, though it has been invoked to explain the origins of skeletal muscle, cartilage, and connective tissue. We have used an analysis of myogenic-precursor cell behavior to develop a model of myogenesis based upon cell lineage commitment. These experiments indicate that primary, secondary, and regenerating skeletal muscle fibers form from myoblasts comrnitted to distinct fast, fast/slow, and slow myogenic lineages and that there is sequential expression of fast and/or slow classes of myosin heavy chain (MHC) within distinct fiber types. The model postulates that myoblast commitment is the basis for restricting MHC expression within primary, secondary, and regenerating fibers to either the fast, slow, or both classes of MHC. Thus this model incorporates two organizing ideas from recent work. First, the many isoforrns of MHC are grouped into two classes (a last and a slow class) both of which contain more than one developmentally regulated isoform. Second, the particular development program for sequential expression of MHC isoforms available to a muscle fiber is dependent on the commitment of the myoblasts from which it forms to distinct myogenic lineages.
51
DEVELOPMENTAL AND TISSUE-SPECIFIC EXPRESSION OF TWO SLOW MYOSIN HC mRNAs IN THE CHICKEN. D. Essig, S. Jakovcic, and P.K. Umeda. U n i v e r s i t y o f Chicago, Chicago IL. T~) slow myosin heavy chain (HC) isoforms (SM-I and SM-2) are d i f f e r e n t i a l l y expressed during the development of avian slow t o n i c muscle f i b e r s . The SM-I HC is predominantly expressed in the embryo, but is replaced by SM-2 HC in the adul t t i s s u e . We have i d e n t i f i e d two cDNA clones, pMHC 15/2 and pMHC 6/1 which encode SM-I and SM-2 HCs, r e s p e c t i v e l y . Sequence a n a l y s i s o f o v e r l a p p i n g regions revealed 75% homology i n d i c a t i n g that each p r o t e i n is encoded by a separate mRNA. Sequences o f the SM-I HC are more s i m i l a r (85-90%) to the mmnmalian slow s k e l e t a l / c a r d i a c ~ HC suggesting i t may be the avian homologue. In c o n t r a s t , tne SM-2 HC does not bear s t r i k i n g homology (75-80%) to other c h a r a c t e r i z e d myosin HC mRNAs anO may be a "slow t o n i c " HC. SI nuclease mapping o f RNA i n d i c a t e d t h a t n e i t h e r SM-I or SM-2 HCs were the major HC mRNA species in cardiac t i s s u e s . Thus, there is a g r e a t e r d i v e r s i t y of slow and cardiac myosin HCs in the chicken in comparison to the mm~al. Using cDNAs f o r slow and f a s t myosin HCs, we examined expression of the mRNAs during e a r l y development. SM-I and SM-2 HC mRNAs were expressed in myotubes derived from primary but not in secondary generation myotubes where o n l y f a s t embryonic HC mRNAs were detected. However, the r a t i o o f SM-I:SM-2 in the primary lineage was several f o l d lower than t h a t observed in v i v o . T h e r e f o r e , whi l e the expression of the slow myosin HC mRNAs is i n t r i n s i c to the F l y l i n e a g e , the r a t i o o f SM-I:SM-2 in vivo must be modulated by additional factors.
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49
AUTONOMOUS DIFFERENTIATION OF CARDIAC MYOBLASTS IN VITRO. D. Bader, T. Schultheiss, H. Holtzer, F. Wachtler. Dept. of Cell Biology and Anatomy, Cornell University Medical College, NY, NY. Dept. of Anatomy, University of Pennsylvannia, Philadelphia, PA. The central portion of blastoderms of the chicken embryo was dissociated into single cells and cultured at high or low density for periods up to five weeks. Clusters of rhythmically beating cells were first observed on the forth day of culture. Cultures were fixed and screened for the presence of sarcomeric myosin heavy chains with a monoclonal antibody which recognizes all sarcomeric myosins and monoclona) antibodies specific for atria), ventricular or conduction system isoforms. In cultures plated both at an initially high or low density cells expressing sarcomeric myosin could be found either as single cells or as clusters. Only in those cultures plated at an initially high density were atrial and ventricular specific isoforms detected, whereas cardiac myocytes in low density cultures as well did not express myosin heavy chains reactive with our tissue specific antibodies. No cells containing conduction system specific myosin isoforms were ever seen in any of our cultures. Addition of TPA during the initial period of culture ( 41 day) prevented the emergence of cardiac myocytes as determined by the absence of beating ceils and sarcomeric myosin heavy chain positive cells. Adding TPA after the onset of cardiac myogenesis did not alter the expression of myosin heavy chains, We conclude that cardiac myoblasts differentiate and diversify in the absence of heart morphogenesis.
50
THE CELLULAR BASIS FOR COMMITMENT IN SKELETAL MUSCLE AND FOR DIVERSITY OF MYOSIN HEAVY CHAIN EXPRESSION IN AVIAN DEVELOPMENT. F.E. Stockdale, ~].B. Miller. Department of Medicine/Oncology Stanford University Medical School, Stanford, California. Diversification of cell types in the embryo is attributed to differences in commitment among the precursor cells that produce different cell types. This concept has not been used to explain the origins of different skeletal muscle fiber types, though it has been invoked to explain the origins of skeletal muscle, cartilage, and connective tissue. We have used an analysis of myogenic-precursor cell behavior to develop a model of myogenesis based upon cell lineage commitment. These experiments indicate that primary, secondary, and regenerating skeletal muscle fibers form from myoblasts comrnitted to distinct fast, fast/slow, and slow myogenic lineages and that there is sequential expression of fast and/or slow classes of myosin heavy chain (MHC) within distinct fiber types. The model postulates that myoblast commitment is the basis for restricting MHC expression within primary, secondary, and regenerating fibers to either the fast, slow, or both classes of MHC. Thus this model incorporates two organizing ideas from recent work. First, the many isoforrns of MHC are grouped into two classes (a last and a slow class) both of which contain more than one developmentally regulated isoform. Second, the particular development program for sequential expression of MHC isoforms available to a muscle fiber is dependent on the commitment of the myoblasts from which it forms to distinct myogenic lineages.
51
DEVELOPMENTAL AND TISSUE-SPECIFIC EXPRESSION OF TWO SLOW MYOSIN HC mRNAs IN THE CHICKEN. D. Essig, S. Jakovcic, and P.K. Umeda. U n i v e r s i t y o f Chicago, Chicago IL. T~) slow myosin heavy chain (HC) isoforms (SM-I and SM-2) are d i f f e r e n t i a l l y expressed during the development of avian slow t o n i c muscle f i b e r s . The SM-I HC is predominantly expressed in the embryo, but is replaced by SM-2 HC in the adul t t i s s u e . We have i d e n t i f i e d two cDNA clones, pMHC 15/2 and pMHC 6/1 which encode SM-I and SM-2 HCs, r e s p e c t i v e l y . Sequence a n a l y s i s o f o v e r l a p p i n g regions revealed 75% homology i n d i c a t i n g that each p r o t e i n is encoded by a separate mRNA. Sequences o f the SM-I HC are more s i m i l a r (85-90%) to the mmnmalian slow s k e l e t a l / c a r d i a c ~ HC suggesting i t may be the avian homologue. In c o n t r a s t , tne SM-2 HC does not bear s t r i k i n g homology (75-80%) to other c h a r a c t e r i z e d myosin HC mRNAs anO may be a "slow t o n i c " HC. SI nuclease mapping o f RNA i n d i c a t e d t h a t n e i t h e r SM-I or SM-2 HCs were the major HC mRNA species in cardiac t i s s u e s . Thus, there is a g r e a t e r d i v e r s i t y of slow and cardiac myosin HCs in the chicken in comparison to the mm~al. Using cDNAs f o r slow and f a s t myosin HCs, we examined expression of the mRNAs during e a r l y development. SM-I and SM-2 HC mRNAs were expressed in myotubes derived from primary but not in secondary generation myotubes where o n l y f a s t embryonic HC mRNAs were detected. However, the r a t i o o f SM-I:SM-2 in the primary lineage was several f o l d lower than t h a t observed in v i v o . T h e r e f o r e , whi l e the expression of the slow myosin HC mRNAs is i n t r i n s i c to the F l y l i n e a g e , the r a t i o o f SM-I:SM-2 in vivo must be modulated by additional factors.
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