Ronald B. Young and Ronald E. Allen Fibers ...

Transitions in Gene Activity during Development of Muscle Fibers Ronald B. Young and Ronald E. Allen J ANIM SCI 1979, 48:837-852.

The online version of this article, along with updated information and services, is located on the World Wide Web at: http://jas.fass.org/content/48/4/837

www.asas.org

Downloaded from jas.fass.org by guest on July 10, 2011

TRANSITIONS

IN G E N E A C T I V I T Y D U R I N G OF M U S C L E FI B E R S a

DEVELOPMENT

Ronald B. Young b'c'd and Ronald E. Allen b'd

Michigan State University, East Lansing SUMMARY

One of the fundamental aspects of skeletal muscle differentiation is activation of the gene programs coding for muscle-specific proteins. This transition encompasses gene products both directly and indirectly involved in skeletal muscles' contractile and developmental activities. This review article therefore examines unique gene transitions in myofibrillar proteins, sarcoplasmic reticulum, acetylcholine receptors, energy metabolism, cell membrane fusion and nucleic acid metabolism. In nearly all eases there is direct evidence that muscle differentiation is accomplished by synthesis of gene products unique to muscle fibers rather than by stimulation of genes that are already active in replicating myogenic precursor cells and nonmuscle cells. (Key Words: Gene Transitions, Myofibrillar Proteins, Membrane Functions, Fusion, Metabolism.) INTRODUCTION

One of the primary goals of animal science is to increase the deposition of myofibrillar proteins in the skeletal muscle of meat animals. The proteins composing the myofibril make up more than half the total protein content of muscle tissue, and an u n d e r s t a n d i n g of the factors t h a t r e g u l a t e accretion of these proteins is of p a r a m o u n t i m p o r t a n c e . D i f f e r e n t i a t i o n of r e p l i c a t i n g , precursor myogenic cells into myofibril-laden,

mature muscle fibers is the result of activation of some new genes as well as suppression of others. The purpose of this article is to review the musclespecific changes in gene expression that take place during muscle cell development, with emphasis on the myofibrillar proteins. Because a limited number of investigations have been conducted at the biochemical level with the goal of u n d e r s t a n d i n g muscle-specific gene p r o d u c t metabolism for the betterment of meat animal production, it is hoped that this review of basic studies in muscle fiber development will stimulate new efforts toward such a goal. The development and growth of functional, contractile muscle fibers from replicating precursor cells can be divided for discussion purposes into a series of stages (figure 1). Generally, the stages correspond to myogenic cell proliferation and protein accretion in muscle fibers; however, it should be kept in mind that muscle tissue d e v e l o p m e n t and g r o w t h are gradual, progressive processes and not a sequence of discrete, synchronized events as may be implied by figure 1. Once a replicating myogenic cell has undergone the proper transitions that cause it to cease proliferation, new gene programs are initiated that result in expression of full myogenic potential. This review will examine these unique activities under the two major headings of (1)gene products directly involved in skeletal muscles' contractile f u n c t i o n , s u c h as m y o f i b r i l l a r p r o t e i n s , sarcoplasmic reticulum and acetylcholine receptors, and (2) gene transitions that indirectly facilitate the c o n t r a c t i l e or d e v e l o p m e n t a l processes, i n c l u d i n g m o d u l a t i o n of energy metabolism, cell membrane fusion and nucleic acid metabolism. Because the myofibrillar proteins compose the largest protein fraction in skeletal muscle, and because either enhanced or more efficient deposition of myofibrillar proteins is desirable in meat animals, emphasis will be placed primarily on the gene programs regulating the myofibrillar protein component of skeletal muscle.

"Michigan Agricultural Experiment Station Article No. 8444. This effort was supported in part by Michigan Agricultural Experiment Station Project Numbers 1241, 1265 and 1280; Biomedical Research Support Grants from the College of Agriculture and Natural Resources and the College of Osteopathic Medicine; and research grants from the Muscular Dystrophy Association of America and the Michigan Heart Association. The authors are grateful to Drs. R. A. Merkel and A. M. Pearson for their helpful suggestions. bDepartment of Animal Husbandry. CDepartment of Biomechanics. dDepartment of Food Science and Human Nutrition. 837 JOURNAL OF ANIMAL SCIENCE, Vol. 48, No. 4- (1979) Downloaded from jas.fass.org by guest on July 10, 2011

838

YOUNG AND ALLEN DIFFERENTIATION AND GROWTH OF SKELETAL MUSCLE

Embryonic Development of all Myogenic Precursors

Satellite Cells (SC)

--~ Proliferation of ~" f Myogenic Precursor~" Cells (PMb)

Cessation of Proliferation and Concomitant Transition of PMb to Mb

Myogenic Cell Proliferation

I m

Activation of Muscle FiberSpecific Gene Programs (Mb or MT)

Accretion of Muscle Fiber Gene Products

Maturity and Operation of Gene Programs at 'Maintenance Levels

Figure 1. A general scheme showing the interrelationship between myogenic cell proliferation and accretion ofgene products unique to musclefibers. Cell types in parenthesis are designated according to the ceil'lineagetheory of Abbott et al., (1974) and are defined as follows: Presumptive Myoblast (PMb), a replicating cell within the myogenic lineage that cannot fuse or synthesize myofibrillar contractile proteins; Myoblast (Mb), a mononucleated myogenic cell that can elaborate musclefiber-specificgene products and can fuse with other Mb, but cannot proliferate; Myotube (MT), a multinucleated cell, formed by myoblast fusion, that has the same metabolic options as a Mb; Satellite Cell (SC), a quiescent mononucleated cell within the myogenic lineage that is normally responsible for adult muscle regeneration and that, after activation to proliferate, is indistinguishable from a PMb. The dashed line between "Embryonic Development of all Myogenic Precursors" and "Satellite Cells" indicates that a small number of myogenic cells (i.e., Satellite Cells) are always found in adult skeletal muscle between the basement membrane and the sarcolemma; however, the mechanism ensuring their presence is not known. GENE PROGRAMS DIRECTLY INVOLVED IN CONTRACTILE ACTIVITY

Myofibrillar Proteins (Myosin and Actin). Much of the research on myogenesis has centered around the process of myoblast fusion into multinucleated myotubes. This emphasis likely results from the impressive nature of the fusionassociated changes in m o r p h o l o g y and the concomitant elaboration of the myofibrillar proteins. However, because fusion and myofibrillar protein synthesis can be temporally dissociated from each other in developing skeletal muscle (discussed below), and because it is clear that fusion is not required for the functioning of other contractile tissues such as cardiac and smooth muscle, the actual process of myoblast fusion is considered supportive or facilitative in skeletal myogenesis and will be considered later.

All cell types are capable of somemovement or motility, and at least two types of contractile systems are k n o w n . The c o n s t i t u t i v e (or cytoplasmic) contractile system is the contractile component responsible for such intracellular processes as cytokinesis, endocytosis, exocytosis, a n d p s e u d o p o d i a f o r m a t i o n ; w h e r e a s the myofibrillar contractile system is composed of the more highly ordered contractile protein aggregates that make up individual sarcomeres and myofibrils in skeletal and cardiac muscle. When a myogenic precursor cell ceases replication (figure 1), the gdnes encoding the information for the myofibrillar contractile system are expressed. The first evidence suggesting that myofibrillar contractile protein synthesis is the result of activation of new genes was obtained by Holtzer et al. (1957). No cross-striations could be found with anti-myosin in chicken embryo somites


GENE TRANSITIONS IN MUSCLE p r i o r to stage 16; however, m o n o n u c l e a t e d myoblasts in the brachial myotomes of stage 16 to 23 somites were easily identified by staining witl~ antibodies against adult chicken skeletal myosin. Although immunological techniques and purification of myosin for use as an antigen have improved drastically over the years, the basic conclusion from the studies of Holtzer et al. (1957) has been substantiated in considerable detail. Extracts from replicating presumptive myoblasts (PMb) arrested in metaphase, for example, do not react immunologically with antibodies to skeletal muscle myosin. However, within several hours following the mitosis which results in formation of a myoblast (Mb) from PMb, myosin can be detected by anti-myosin antibodies (Holtzer, 1970). These data are consistent with activation of skeletal muscle m y o f i b r i l l a r m y o s i n genes following a particular cell division and support the quantal mitosis concept of the myogenic lineage (Holtzer, 1970). Indeed, most of the known changes associated with skeletal muscle differentiation are likely highly coordinated and can be traced back to basic gene activation within a matter of hours following a particular mitosis. Because these two immunological investigations ( H o l t z e r et al., 1957; H o l t z e r , 1970) were conducted with antibodies to myofibrillar myosin, and because no skeletal muscle myosin was ever detected with these antibodies in replicating PMb or other nonmuscle cells, it was concluded that these replicating cells contained no myosin at all. These studies were conducted prior to discovery of the constitutive contractile system in all cell types (reviewed by P o l l a r d and Weihing, 1974); therefore, the fact that anti-skeletal-muscle myosin did not react with precursor cells is, in retrospect, evidence that new, immunologically specific myofibrillar contractile protein genes are activated during myogenesis. The ability to separate myogenic cells into individual cell compartments either by preventing the transition of PMb to Mb (Cohen et al., 1977; Holtzer et al., 1975; Stockdale et al., 1964) or by halting Mb prior to fusion (Holtzer et al., 1975; 1976; Paterson and Strohman, 1972; Sanger, 1974; Van der Bosch et al., 1972) has enhanced identification of the specific metabolic programs in these two cell types. Antibodies to adult skeletal muscle light meromyosin (anti-LMM) bind to Abands of myofibrils in myoblasts prevented from f u s i n g a n d to A - b a n d s in m u l t i n u c l e a t e d myotubes. Additionally, anti-LM M forms a single precipitin band in Ouchterlony immuno-diffusion tests with .6 M KCI extracts of mature myotubes

839

(Chi et al., 1975a; Cohen et al., 1977; Fellini and Holtzer, 1976). There is no binding, however, of anti-LMM to (1) the cytoplasm, nuclei or cell surface of either mononucleated myoblasts or multinucleated myotubes, (2) the cell surface, nuclei or cytoplasm of presumptive myoblasts, fibroblasts, bromodeoxyuridine-suppressed presumptive myoblasts, chondroblasts, or several o t h e r n o n m u s c l e cell types, or (3) to the microfilaments subtending the sarcolemma in multinucleated myotubes (Chi et al., 1975a; Fellini and Holtzer, 1976). The microfilaments are c o m p o s e d of actin as evidenced by heavy meromyosin decoration (Ishikawa et al., 1968)and therefore might be expected to have constitutive myosin associated with them. In addition, the quantity of myosin associated with these cellular structures was sufficient to be detected by the indirect immunofluorescent antibody technique, since c o n c e n t r a t e d .6 M K C l - e x t r a c t s from fibroblasts, PMb, chondroblasts, brain and liver did not form bands in Ouchterlony immunodiffusion tests (Fellini and Holtzer, 1976). These data are consistent with the notion that the myosin found in thick filaments of skeletal muscle myofibrils is unique to skeletal muscle and is, therefore, different from the constitutive myosin molecule t h a t is associated with cell surfaces, m i c r o f i l a m e n t s , nuclei or c y t o p l a s m . These immunological differences suggest that myofibrillar myosin composing thick filaments in skeletal muscle is the product of a gene that is inactive in replicating presumptive myoblasts or other nonmuscle cells. The primary function of the myosin molecule during contraction or motility is to interact with actin filaments and, upon hydrolysis of ATP, to undergo a confromational change that results in movement of myosin relative to the actin filament. Myosin, t h e r e f o r e , is an enzyme for A T P hydrolysis, and the ATPase activity of native myosin from a variety of cell types has been assessed in an attempt to define unique functional aspects of the protein and to relate ATPase activity to light chain composition. In general, constitutive myosin ATPase activity is lower than skeletal muscle myosin ATPase, and the degree of skeletal muscle actin activation of constitutive myosin ATPase is less than skeletal muscle actin activation of skeletal muscle myosin ATPase (Adelstein et al., 1972; Chi et al., 1975a,b; Ostlund et al., 1974; Pollard and Weihing, 1974; Rubenstein et al., 1974). These., data suggest differences among tissues in the c o m p o s i t i o n of myosin, a n d structural analyses have confirmed this possibility.


840

YOUNG AND ALLEN

The individual polypeptides composing myosin can be dissociated in the presence of denaturing agents such as sodium dodecyl sulfate (SDS) and the composition of the molecule electrophoretically studied. While such investigations have failed to reveal major size differences in myosin heavy chains (MHC) between adult skeletal muscle and a variety of eukaryotic nonmuscle cells (Burridge and Bray, 1974), the molecular weight distribution of the light chains of myosin (MLC) has been found to vary in a manner that is consistent with expression of different genes in nonmuscle cells and skeletal muscle. For example, myosin purified from embryonic chicken multinucleated myotubes contains two sets of light chains of 25,000 and 18,000 daltons, whereas myosin from PMb and fibroblasts contains light chains of 16,000 and 14,000 daltons (Chi et al., 1975a, b; Rubinstein et al.,1974). Qualitatively similar results have also been reported in rat muscle cultures (Yablonka and Yaffe, 1976, 1977). These distinctive differences in MLC suggest that the myosins are coded by separate sets of structural genes. Indeed, there may be several different MLC genes because distinctive MLC patterns have been reported for fast, slow, cardiac and smooth muscle myosins, as well as for platelet, fibroblast, chondroblast, presumptive myobtast and BrdUsuppressed myogenic cell myosin (Burridge and Bray, 1975; Chi et al., 1975a; Cohen et al., 1977; Kendrick-Jones, 1973; Lowey and Risby, 1971; Ostlund et al., 1974; Sarker et al., 1971; Weeds, 1969). Changes in relative quantity of MLC! (23,000 daltons) and MLC2 (17,000 daltons) m R N A in developing rat myoblast cultures correlates well with relative changes in the rates of synthesis of these MLC peptides; however, MLC3 (15,000 daltons) synthesis may be subject to posttranscriptional modulation (Yablonka and Yaffe, 1976, 1977). M LC composition may also vary with developmental age. While myosin purified from embryonic chicken breast muscle or embryonic breast muscle cell cultures apparently is homogeneous and contains only two sets of light chains (Burridge and Bray, 1975; Chi et al., 1975b), the myosin in myofibrils isolated from adult fast muscle contains three sets of light chains and has been proposed to be made up of a mixture of two types of native myosin molecules (Burridge and Bray, 1975; Sarkar, 1972). The presence of a distinctive light chain pattern for a variety of cell types suggests functional differences, and although the full significance of this variation is not yet known, a plausible explanation has recently been suggested. The

20,000 dalton MLC from blood platelets can be p.hosphorylated (Adelstein et al., 1973), and this phosphorylated myosin, when reeombined with skeletal muscle F-actin, has a higher actinactivated ATPase activity than dephosphorylated myosin (Adelstein and Conti, 1975). These data suggest that a p h o s p h o r y l a t i o n - d e p h o s p h o r y lation cycle may contribute to regulation of myosin activity and, therefore, to the contractile activity in certain cells containing the constitutive contractile system. A similar mechanism has been proposed for regulation of contraction in smooth muscle (Sobieszek, 1977; Small and Sobieszek, 1977). Smooth muscle cells contain MLC kinase that can phosphorylate a specific serine residue on the 20,000 dalton MLC. Phosphorylated smooth muscle myosin has a higher actin-activated ATPase activity than the nonphosphorylated species, suggesting that phosphorylation enhances the interaction between myosin and actin. Not unexpectedly, smooth muscle cells also contain a phosphatase enzyme capable of dephosphorylating MLC, and, therefore, inactivating the c o n t r a c t i l e p r o c e s s ( M o r g a n e t a l . , 1976; Sobieszek, 1977). Furthermore, MLC kinase is activated by the same Ca 2§ concentration that normally activates skeletal muscle contraction. Thus, while contraction in both smooth and skeletal muscle is regulated by changes in Ca 2§ concentration, different biochemical processes are involved. The role of myosin phosphorylation during muscle cell differentiation has also been studied in proliferating myogenic cells in culture ( S c o r d i l i s a n d A d e l s t e i n , 1977). W h i l e phosphorylation of the 20,000 dalton MLC in these replicating myogenic cells is a prerequisite for actin activation of myosin ATPase, the endogenous MLC kinase responsible for MLC p h o s p h o r y l a t i o n does not require Ca :+ for activity. These results suggest that, in addition to heterogeneity in the number of structural genes coding for both MHC and MLC, different cell types may possess a variety of mechanisms for activating their intracellular contractile processes. As indicated earlier, only slight differences have been observed in the electrophoretic mobility of MHC from various tissues. Other studies on the susceptibility of myosin to proteolytic enzymes and reaction with 2-nitro-5-thiocyanobenzoic acid, however, have p r o v i d e d evidence for differences in the primary structure of MHC in different tissues (Burridge and Bray, 1975). A l t h o u g h d i f f e r e n c e s in s u s c e p t i b i l i t y to p r o t e o l y s i s could be i n t e r p r e t e d as subtle alterations in primary, secondary or tertiary


GENE TRANSITIONS IN MUSCLE structure (possibly as a result of m o d i f i e d interaction between different light chains and the same set of heavy chains), different peptide maps obtained after eyanylation of myosins from different tissues can only be e x p l a i n e d by differences in the primary structure of MHC because 2-nitro-5-thiocyanobenzoic acid specifically cleaves peptides at cysteine residues. Burridge and Bray (1975) have found that several specific, primary types of myosin peptide patterns can be obtained from chicken breast, heart, gizzard, platelet and brain, and that peptide patterns from other tissues match these patterns either singly or in combination. By examination of peptide patterns from both intact skeletal myosin and its purified proteolytic subfragments, it could be deduced that most of the large cyanylation cleavage products appeared to come from the rod portion of the myosin molecule. More recently, Patrinou-Georgoulas and John (1977) have shown that MHC purified from embryonic muscle or cultured muscle cells can be isoelectrically focused i n t o t w o d i s t i n g u i s h a b l e c o m p o n e n t s in approximately equal amounts. This observation could be explained either by a homogeneous population of native myosin molecules in which the two c o m p o n e n t heavy chains were not identical, or by equal quantities of two populations of native myosin each of which had identical heavy chains. In any case, the results discussed in this section suggest tissue-specific differences in both the globular region of myosin (which possesses the ATPase activity) and the rod portion of myosin (which is involved in packing into thick filaments). The most likely explanation for these observations is that the eukaryotic genome contains a number of structural genes which code for the appropriate tissue-specific MHC and MLC. The second most abundant contractile protein in skeletal muscle myofibrils is actin. Actin has the ability to form long polymers in the presence of A T P and physiological ionic conditions, and it composes the thin filaments in the myofibril and the "microfilaments" of the constitutive contractile protein system. Actin has been identified in a large number of cells and tissues (Pollard and Weihing, 1974), and it now seems apparent that actin is ubiquitous to all eukaryotes. Because of structural and physiological similarities in actins from all cell types examined, and because of difficulties in raising antibodies to native actin, the questions have been raised whether all actins evolved from the same structural gene and whether the genome of a given species contains only one actin gene whose p r o d u c t s are utilized for b o t h the

841

constitutive and the myofibrillar contractile systems. It now seems certain that there is more than one structural gene for actin. For example, the actin isolated from chicken brain has a slightly different m o b i l i t y d u r i n g e l e c t r o p h o r e s i s in polyacrylamide gels containing urea and SDS than actin isolated from chicken skeletal muscle (Storti and Rich, 1976), and peptide maps of these p r o t e i n s e x h i b i t slightly different p a t t e r n s (Gruenstein and Rich, 1975; Storti and Rich, 1976; S t o r t et al., 1976). T h e s e e l e c t r o p h o r e t i c differences result from unequal primary structure of the structural genes for these proteins rather t h a n p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n of the synthesized peptides (Storti and Rich, 1976). Total messenger RNA from brain and from muscle tissue was extracted and used to direct protein synthesis in cell-free protein-synthesizing assays. When a u t o r a d i o g r a m s of the in v i t r o synthesized p r o d u c t s were c o m p a r e d d i r e c t l y with the electrophoretograms of purified tissue proteins, the difference in electrophoretic mobility of brain compared to muscle was maintained in the cell-free p r o t e i n synthetic p r o d u c t s . Therefore, the possibility that actin in these different tissues was modified subsequent to protein synthesis was eliminated, and it seems likely that brain and muscle actins arise from different structural genes ( S t o r t i and R i c h , 1976). M o r e d e f i n i t i v e experiments have shown that human platelet and human cardiac actins have different amino acid sequences (Elzinga et al., 1976). Transitions in expression of actin isozymes in differentiating skeletal muscle are particularly interesting because of the known alterations in contractile protein metabolism that accompany muscle development. Analysis of purified actin from skeletal muscle by isoelectric focusing has shown that there are at least three isozymes of actin expressed in this tissue (Whalen et al., 1976), Whereas the actin purified from nonmuscle cells, such as presumptive myoblasts and kidney cells, contains predominatly two of the three isozymes, actin purified from well-differentiated myotubes or fetal muscle tissue contains primarily one of the three isozymes. These three actin species have been tentatively designated a, 13 and y-actin. It is not clear at this time whether individual muscle fibers contain all three actin isozymes, or whether only the a-actin is present in muscle fibers and the/3and 3,-actins are contributed by the constitutive contractile system microfilaments in the fraction of nonmuscle cells that almost always contaminate muscle cell cultures. Neither possibility has been unequivocally ruled out. Furthermore,


842

YOUNG AND ALLEN

interpretation of the peptide map and electrophoretic analysis of ~/ctins isolated from brain and skeletal muscle (Storti and Rich, 1976; Storti et aL, 1976) is rendered more difficult because the results could be accounted for either by a d d i t i o n a l a c t i n i s o z y m e s or by d i f f e r e n t proportions of the a-, B- and 3,-actin isozymes already known. While the evidence discussed above seems c o n c l u s i v e t h a t m u s c l e d e v e l o p m e n t is accompanied by activation of new actin and myosin genes, immunological data on other c o n t r a c t i l e p r o t e i n s a r e n o t so c l e a r - c u t . Antibodies raised against fibroblast actin, for example, may react with skeletal muscle actin (Lazarides and Weber, 1974). In addition, whereas antibodies against skeletal muscle tropomyosin have been used to localize thin filaments in f i b r o b l a s t s ( L a z a r i d e s , 1975), o t h e r s have indicated that fluorescein-labeled antit r o p o m y o s i n does not bind to p r e s u m p t i v e m y o b l a s t s ( R u b i n s t e i n et al., 1974). These uncertainties may be resolved when m o r e information, is gained about differences in the primary structure of isozymes and subunits (in the case of tropomyosin) of these proteins. All observations described to this point are consistent with multiple genes for myosin and actin in the eukaryotic genome. The presence of multiple genes for myosin and actin within a given genome suggests that individual cells are able to exercise independent control over the synthesis of mRNA for these proteins for different cellular functions and, therefore, to regulate the quantity of protein available for each function. This alleviates the necessity for a cell to undergo complex modulation of a single gene for all these functions. The key questions with regard to the muscle fiber-specific contractile protein genes in meat animals are as follows: (1) what conditions selectively activate these genes during muscle development, (2) why are the products of these genes accumulated in muscle fibers to such an e n o r m o u s e x t e n t compared to the contractile proteins of the constitutive contractile system in nonmuscle tissues, and (3) what factors or conditions could be used to modify the activity of the myofibrillar protein genes so that more extensive or more efficient deposition of muscle proteins occurs? Answers to these questions represent key areas in understanding regulation of myofibrillar protein metabolism in meat-producing animals. S a r c o p l a s m i c R e t i c u l u m . The myofibrillar proteins, which make up the largest protein fraction in muscle tissues and whose interactions

lead to muscle contraction, are the most widely studied component of skeletal muscle. However, at least two b i o c h e m i c a l processes o t h e r t h a n movement of thick and thin filaments relative to each other are integral parts of the overall contractile event. These include acetylcholine (ACh)-induced depolarization of the muscle fiber membrane during nerve impulse transmissioh and regulation of intracellular Ca 2§ concentration by the sarcoplasmic reticulum. When the neurotransmitter, ACh, is released from nerve endings it interacts with the muscle fiber sarcolemma and thereby depolarizes the muscle fiber membrane. Free ACh is quickly hydrolyzed by aeetylcholine esterase (ACHE) assembled in the ACh receptor, but the wave of depolarization induced by ACh is carried deep inside the muscle fiber by the transverse tubules where it causes release of stored Ca 2+ from the sarcolasmic reticulum. The released Ca 2+ interacts with the troponin complex to cause movement of tropomyosin toward the groove of the thin filament, and this movement allows the myosin head to interact with actin so that contraction occurs. Once the wave of depolarization is complete, the sarcoplasmic reticulum reaccumulates Ca 2§from the cytoplasm of the muscle cell, thus i n h i b i t i n g the c o n t r a c t i l e process (Mannherz and Goody, 1976). The sarcoplasmic reticulum membrane has been well characterized in its function, Ca 2§ transport ability, ultrastrueture, protein composition and lipid composition (Boland et aL, 1974; MacLennan and Holland, 1975; Martonosi et al., 1972; Sarzala et al., 1975; Tillack et al., 1974). Briefly, the m e m b r a n e c o n t a i n s an A T P a s e e n z y m e (asymmetrically oriented toward the cytoplasmic side of the membrane), calsequestrin (for internal Ca 2+ storage in relaxed muscle), a low molecular weight proteolipid, and a high-affinity calcium binding protein. These two latter proteins are localized on the inner side of the sarcoplasmic reticulum membrane (MacLennan et al., 1971; 1972; MacLennan and Wong, 1971; Meissner, 1975; Ostwald and MacLennan, 1974; Stewart and MacLennan, 1974). Functional development of sarcoplasmic reticulum has been quantitatively studied in cultured muscle cells (Lough et al., 1972), and both the ATPase of the sarcoplasmic reticulum and its a b i l i t y to s e q u e s t e r Ca 2+ d u r i n g m u s c l e d i f f e r e n t i a t i o n has been f o u n d to d e v e l o p gradually over several days. Both activities seemed to be present at low, but detectable levels before i n i t i a t i o n of massive, muscle-specific gene


GENE TRANSITIONS IN MUSCLE

843

transitions. To determine if these increases in sarcoplasmic reticulum components represented a quantitative activation of genes already being transcribed to a limited extent in presumptive myoblasts, or if they represented activation of entirely new genes, cultured skeletal muscle cells were carefully scrutinized with antibodies specific for two of the proteins of the sarcoplasmic reticulum ( H o l l a n d and M a c L e n n a n , 1976; J o r g e n s e n et a l . , 1977; Z u b r z y c k a a n d MacLennan, 1976). Quantitative increases in the amounts of both ATPase and calsequestrin appear to coincide with increases in other muscle proteins during differentiation, and replicating fibroblasts do not contain immunologically detectable levels of ATPase or calsequestrin. Therefore, the protein components of the sarcoplasmic reticulum seem to be unique to muscle fibers ( H o l l a n d and MacLennan, 1976; Zubrzycka and MacLennan, 1976), but it should be kept in mind that the activity of both the constitutive contractile system and the skeletal muscle myofibrillar contractile system are regulated by Ca 2+ (although probably by different biochemical mechanisms as discussed earlier). All cell types, then, must process a means to carefully regulate the c o n c e n t r a t i o n of intracellular Ca 2".

receptor sites in the cell membrane probably reflects the amount of time required for depletion of the supply of preassembled receptor sites. F u r t h e r m o r e , i n h i b i t i o n of A T P synthesis immediately suppresses the appearance of new receptor sites and suggests that ACh receptor assembly is an energy-requiring process (Hartzell and Fambrough, 1973). Because there is an enormous quantitative increase in the amount of a c e t y l c h o l i n e esterase (ACHE) that can be extracted from muscle cells during the differentiation, and because fibroblasts contain almost undetectable quantities of A C h E (Wilson, et aL, 1973), activation of muscle specific genes for the enzyme is suggested (Fambrough and Rash, 1971; Fischbach eta/., 1971; Fluck and Stro hman, 1973; Paterson and Prives, 1973; Tennyson et al., 1973; Wilson et al., 1973). Three isozymes of ACh have been identified in muscle fibers (Friedel and Johnson, 1977; Wilson et aL, 1969; Wilson et al., 1973), but neither the factors that regulate the relative levels of these three isozymes in skeletal muscle nor the exact function of the individual isozymes is known.

Acetylcholine Receptors. Transmission of a nerve impulse from the motor endplate into the interior of the muscle cell where myofibrils are located is essential for myofibrillar contraction, and development of this specialized ability is an integral part of muscle development. Because the chemical messenger between nerve endings and muscle cells is A C h , events controlling the appearance of ACh receptors in plasmalemma of myogenic cells have been studied. Hartzell and Fambrough (1973) and Fambrough (1974), for example, have demonstrated that ACh receptor density increases at the rate of approximately 35 sites/#m2/hr immediately after initiation of myoblast fusion. These new sites are functional ACh receptors and are evenly distributed over the surface of the myotubes (Fambrough et al., 1974; Hartzell and Fambrough, 1973). ACh receptors also continue to appear on the plasma membranes of cultured myogenic cells for several hours after administration of eycloheximide to block protein synthesis ( F a m b r o u g h , 1974; H a r t z e l l and Fambrough, 1973); therefore, it seems likely that ACh receptors are constructed intracellularly and that completed receptors are then assembled intact into the cell membrane. The fact that there is a significant lag time between inhibition of protein synthesis and the decreased rate of appearance of

A l t e r a t i o n s in E n e r g y M e t a b o l i s m . The primary focus to this point has been on the gene transitions affecting the structural and regulatory components of the contractile apparatus. Several other unique transitions are integral parts of muscle d e v e l o p m e n t , and one of the most important is development of the capacity to provide energy for contraction. Because this energy is derived from hydrolysis of ATP (Taylor, 1972; Tonomura and Oosawa, 1972), muscle differentiation would be expected to accentuate the activity of pathways involved in producing this high energy compound. The glycolytic pathway is one of the major metabolic pathways responsible for producing ATP, and activities of glycogen phosphorylase (Shainberg et al., 1971; Wahrman et aL, 1973), glycogen synthetase (Wahrman et aL, 1973), aldolase (Turner et al., 1974; 1976), phosphoglycerokinase (Hauschka, 1968) and pyruvate kinase (Guguen-Guillouze et aL, 1977) increase several-fold during muscle cell differentiation. In addition, much of the energy reserve in muscle is stored as creatine phosphate, which is synthesized from ATP and creatine by the enzyme, creatine phosphokinase. At least 10 to 15 times more creatine phosphokinase is found in the cytoplasm of muscle fibers than in the cytoplasm of replicating cells (Keller and Nameroff, 1974;

GENE TRANSITIONS INDIRECTLY INVOLVED IN CONTRACTION OR DEVELOPMENT


844

YOUNG AND ALLEN

Lough and Bischoff, 1977; Morris et al., 1976; Shainberg et al., 1971; Tarikas and Schubert, 1974; Turner et al., 1974; 1976). Whether the dramatic increase in activity of enzymes associated with providing a continuous supply of energy for contraction results from specific gene transitions has been investigated in some detail. Most of the enzymes mentioned above are known to exhibit a variety of tissue-specific isozymes. These isozymes are of course the product of different structural genes, and characteristic transitions toward the muscle-specific isozyme patterns have been observed during muscle cell differentiation for aldolase, pyruvate kinase, and creatine phosphokinase (Guguen-Guillouze et al., 1977; Keller and Nameroff, 1974; Lough and Bischoff, 1977; Morris et aL, 1976; Turner et al., 1974, 1976). A c t i v a t i o n of muscle-specific isozymes of key metabolic enzymes seems to be a necessary adjustment of muscle fibers; however, considerable levels of the nonmuscle isozymes remain in fully developed muscle cell cultures. Whether this observation is an artifact of an imperfect cell culture model, or whether full development does not quantitatively suppress the availability for transcription of the nonmuscle isozyme genes is not known. Nevertheless, it is clear that a shift toward the muscle isozyme pattern occurs and that specific gene transitions partially govern the availability of energy for muscle contraction. M y o b l a s t Fusion. It was indicated earlier that much of the research on myogenesis has centered on the process of myoblast fusion, probably because fusion is morphologically unique and because elaboration of muscle-specific changes can be correlated closely with fusion. The purpose of this section is to show that fusion is not an absolute prerequisite for activation of the other muscle fiber-specific gene programs, but rather is one of the many unique events that make up the complex process of muscle cell differentiation. Before a myogenic cell can fuse with a myotube or with another myogenic ceil, the two cells must be in direct contact with each other. Bischoff and Lowe (1974) have shown with the aid of time lapse cinematography that cultured mononucleated myogenic cells move almost constantly and spend approximately three-fourths of their time in contact with a myotube. Initial contact is usually followed by immediate alignment of a myogenic cell parallel to a myotube; however, myogenic cells frequently move back and forth along the myotube for several hours prior to fusion. For cells that have been actively moving along myotubes, the delay

between cessation of myoblast movement and c o n f i r m e d f u s i o n i n t o t h e m y o t u b e s is a p p r o x i m a t e l y 11 minutes. M o r e o v e r , the minimum time lag between the end of mitosis and the earliest onset of fusion was found by Bisehoff and Lowe (1974) to be approximately 5 hr; this agrees closely with e a r l i e r a u t o r a d i o g r a p h i c studies (Bischoff and Holtzer, 1969). The nature of the binding site between fusion competent cells is not known. Treatment of these cells with EDTA (Bischoff and Lowe, 1974) or phospholipase C (Nameroff, 1974) will remove various components from the surface and will prevent fusion (but not adhesion) of the cells. Readdition of the E D T A - e x t r a c t e d material permits fusion to continue, however, readdit[on of the phospholipase C-extracted material actually inhibits further fusion (Bischoff and Lowe, 1974; Nameroff, 1974). The released material from fusion competent cells contains proteins and glycoproteins, but neither the identification of specific components nor the assignment of specific roles to these components has been accomplished. In addition, it is almost certain that Ca ~+ is a b s o l u t e l y r e q u i r e d for p r o p e r binding site interaction, since fusion can be reproducibly blocked at Ca 2§ concentrations of less than 50 #M (Shainberg et al., 1969; Van der Bosch et al., 1972). The ultrastructure of the fusion process has been examined microscopically by a number of workers ( r e v i e w e d by S t r o m e r et al., 1974). T h e plasmalemma of two cells in close apposition to each other simply seem to disappear in several regions. At a number of points the plasmalemma of the myoblast is continuous with that of the myotube, and what appears to be vesicles and tubules resuRing from partial membrane breakdown are found between the cytoplasms of the two cells. E s t a b l i s h m e n t of c o m p l e t e cytoplasmic continuity is very rapid as evidenced by a mosaic-like appearance resulting from g r a d u a l mixing of the f o r m e r m y o b l a s t s ' s c y t o p l a s m ( w h i c h has a h i g h r i b o s o m e concentration) with the cytoplasm of the myotube (Shimada, 1971). In contrast to the documented transitions discussed earlier in myosin and actin genes during muscle development, the specific, macromolecular alteration that results in massive fusion of myogenic cells is completely unknown. Although it might well be suspected that some as yet uncharacterized, specific gene transition causes m y o b l a s t fusion, it could also result f r o m alterations in intracellular conditions unique in muscle cells. Unquestionably, membrane fusion is a widely occurring b i o l o g i c a l process (e.g.,


GENE TRANSITIONS IN MUSCLE e n d o c y t o s i s , e x o c y t o s i s , cell cleavage and membrane assembly), and the intracellular events that result in fusion on a micro-scale in nonmuscle cells could conceivably occur on a larger-scale during muscle development. Because of its prominent role in myogenesis, the fusion process has been implicated as the central event that allows all the other muscle-specific events to occur. This view of myogenesis suggests that the morphological event of membrane fusion somehow activates the internal muscle-specific gene programs. That this does not occur is illustrated by the fact that it is possible to c o m p l e t e l y block m e m b r a n e fusion w i t h o u t preventing subsequent activation of a number of muscle-specific gene programs. Typical experiments of this sort have been carried out in t h e following way using embryonic chicken muscle cell cultures. Extracellular Ca 2+ concentration is reduced below the critical level that permits fusion (Approximately 50#M) either by extensive dialysis of all cell culture medium components or by addition of carefully adjusted levels of EGTA. EGTA has a high affinity for Ca 2§ and, therefore, can be used to buffer Ca 2+ to the desired concentration. Under these conditions fusion of cultured muscle cells is prevented, p r o b a b l y because Ca 2+ is required for proper membrane interaction. Appropriate measurements can then be made to detect the muscle fiber-specific gene product of interest. Use of this approach has led to detection of nearly normal quantities of a number of muscle-specific molecules, such as MHC, actin, ACHE, creatine phosphokinase (CPK), aldolase, SR A T P a s e , calsequestrin, and a s s e m b l e d myofibrils (Allen et al., 1977; Chi et al., 1975b; E m e r s o n and Beckner, 1975; H o l l a n d and MacLennan, 1976; Jorgensen et al., 1977; Keller and Nameroff, 1974; Moss and Strohman, 1976; Paterson and Prives, 1973; Turner et al., 1974, 1976; Vertel and Fischman, 1976). A possible exception to the general pattern has been recently reported by Shainberg and Brik (1978). These workers have suggested that the relationship between fusion and muscle-specific gene activation may be species-specific and have p r e s e n t e d evidence that synthesis of AChE and CPK in rat myoblasts is not activated under any circumstances until fusion has been i n i t i a t e d . In s u m m a r y , muscle cell m e m b r a n e fusion is apparently a reliable marker for normal muscle cell differentiation, but may not be essential for elaboration of muscle fiber-specific gene programs n o r assembly of m y o f i b r i l l a r p r o t e i n s into f u n c t i o n a l myofibrils. The fact t h a t stable,

845

postmitotic, mononucleated myoblasts can be experimentally prepared also suggests that the fusion process itself does not induce myogenic cells to withdraw from the cell cycle as has been suggested (Buckley and Konigsberg, 1974, 1977). This issue, however, remains unsettled. Changes in Nucleic A c i d Metabolism. Emphasis t h u s f a r has b e e n p l a c e d on i n d i v i d u a l d o c u m e n t a t i o n of the known, specific gene transitions that make up muscle differentiation. Activation of these new genes coincides for the most part with cessation of myogenic cell replication. Not unexpectedly, therefore, measurable alterations in the overall metabolism of both RNA and DNA are found to accompany this drastic reorganization of gene expression. In this final section, the extent of overall changes in nucleic acid metabolism and the implications of these alterations will be examined. The first conclusive demonstration that DNA synthesis subsides as a prerequisite to muscle differentiation was made by Stockdale and Holtzer (1961). These workers found that nuclei in mononucleated cells undergoing mitosis are later incorporated into multinucleated myotubes, but that no further DNA synthesis occurred in these nuclei after they had been incorporated into the multinucleated myotubes. These experiments implied that fusion of mononucleated cells into multinucleated myotubes was accompanied by almost complete loss of the ability of nuclei in multinucleated muscle cells to synthesize DNA. These results have since been confirmed by a large number of workers using more direct and quantitative techniques (Okazaki and Holtzer, 1966; O ' N e i l l and S t o c k d a l e , 1972; O'Neill and Strohman, 1969, 1970; Paterson and Strohman, 1972; Stockdale, 1970). P e r h a p s the most d r a m a t i c b i o c h e m i c a l demonstration of cessation of DNA synthesis during muscle-specific gene activation was made by O'Neill and Strohman (1970). These workers pulse-labeled cultures of embryonic muscle cells at different stages of d i f f e r e n t i a t i o n with [3H]Thymidine and measured the percentage decrease in amount of [~H]Thymidine incorporated into partially purified DNA. The percentage d e c r e a s e in [ ~ H ] T h y m i d i n e i n c o r p o r a t i o n coincided almost identically with the percentage decrease in number of nuclei in mononucleated cells due to fusion of these cells to form multinucleated myotubes. Clearly, cessation of cell p r o l i f e r a t i o n and muscle fiber-specific gene activation are closely associated events, and although most evidence would suggest that


846

YOUNG AND ALLEN

cessation of myogenic cell replication is an absolute prerequisite to muscle fiber-specific gene activation, this issue has not been completely settled (Buckley and Konigsberg, 1974, 1977). [3H]Thymidine incorporation into DNA is a direct measure of DNA polymerase activity in these cultures, and it has been suggested that the decrease in DNA polymerase activity accompanying myoblast fusion results specifically from a reduction in the quantity of DNA polymerase enzyme present in the nuclei of the differentiating myogenic cells rather than from an inhibition of the activity of existing DNA potymerase (O'Neill and Strohman, 1969, 1970; Stockdale, 1970). Although it has been rigorously shown that DNA synthesis is almost totally inhibited in the nuclei of myotubes (O'Neill and Stockdale, 1972; O'Neill and Strohman, 1969, 1970; Okazaki and Holtzer, 1966; Paterson and Strohman, 1972; Stockdale, 1970; Stockdale and Holtzer, 1961), it was unclear for several years whether this decrease in DNA synthesis applied only to the synthetic events required for semiconservative replication of DNA, or whether both replicative and repair synthesis were inhibited once nuclei had fused into m u l t i n u c l e a t e d myotubes. S t o c k d a l e (1971) irradiated muscle cell cultures with ultraviolet light at levels adequate to cause formation of significant levels of pyrimidine dimers and found that myotube cultures retained their ability to excise their dimers and to replace them with [3H]Thymidine. Repair DNA synthesis, however, occurred in the myotube nuclei at only half the rate that it did in proliferating myoblasts. Similar results concerning the ability of nonreplicating myotube nuclei to effect repair DNA synthesis have been obtained by using univalent alkylating agents to induce mutations (Hahn et al., 1971). Together the results of Hahn et aL, (1971) and Stockdale (1971) indicate at least four conclusions concerning DNA synthesis in myotube nuclei: (!) the factors controlling semiconservative D N A replication do not control D N A repair; (2) differentiated skeletal muscle cells can phosphorylate and incorporate [3H]Thymidine i n t o D N A w i t h i n m y o t u b e n u c l e i ; (3) a polymerizing enzyme must be present in sufficient quantities for this DNA repair synthesis to take place; and (4) the ability to repair chemically altered bases in DNA is partially lost during differentiation. Whether DNA repair synthesis requires reversal of the controls prohibiting replicative synthesis or whether it requires the maintenance or induction of a separate repair system has not been determined.

In addition to termination of DNA synthesis, muscle differentiation is also accompanied by a decrease in the a m o u n t of nuclear D N A dependent, RNA polymerase activity (Herrmann et al., 1970; Marchok, 1966; Marchok and Wolff, 1968; Yaffe and Fuchs, 1967). Marchok and Wolff (1968) have shown that this decrease in total RNA polymerase activity results from a true decrease in the quantity of enzyme that can be extracted from the nuclei of myotubes rather than inhibition of activity of existing enzyme molecules. It is not known, however, if the unique sequence of events leading to muscle differentiation causes a suppression of the quantity of RNA polymerase found in the nucleus, or if different isozymes of R N A polymerase are induced during muscle development to more efficiently transcribe the smaller n u m b e r of specific gene sequences available for transcription (see below) in welldifferentiated muscle fibers. In view of the large quantities of myofibrillar protein mRNA that must be transcribed in the muscle cell nucleus (i.e., myofibrillar proteins make up over half of the total protein content of muscle), it would seem likely that these muscle nuclei possess a mechanism whereby RNA polymerase preferentially interacts with the myofibrillar protein genes. The nature of this mechanism is not known, however, its specificity most likely resides with the specific gene sequences involved rather than with the enzyme which transcribes them. Earlier in this manuscript, it was stated that differentiation of replicating, precursor myogenic cells into myofibril-laden, mature muscle fibers is the result of activation of some new genes as well as suppression of others. This fact recently has been thoroughly documented in a study by Devlin and Emerson (1978) in which changes in the synthesis rates of a large number of peptides were examined throughout myogenesis. These alterations ranged from 500-fold increases for some species to manyfold decreases for others. It is difficult in many instances to d i s t i n g u i s h between q u a l i t a t i v e stimulation (or inhibition) of the activity of specific gene sequences and quantitative modulation of these genes. That is, in certain instances it may be possible that a gene is temporarily quantitatively suppressed by the unique conditions to which the cell is exposed, but it is capable of immediate e x p r e s s i o n should e n v i r o n m e n t a l c o n d i t i o n s change. The presence of even one m R N A molecule/cell means that the gene is "active," although this activity is obviously very low. In contrast, many heterologous tissue-specific gene programs (e.g., hemoglobin, ovalbumin) are


GENE TRANSITIONS IN MUSCLE qualitatively and irreversibly inactivated in other highly specialized differentiated tissues, such as skeletal muscle. These same tissues may contain many other tissue-specific genes that are extremely active. Differentiated muscle cells, for example, contain up to 10,000 copies per nucleus of the mRNA that codes for MHC (John et al., 1977; P a t e r s o n and Bishop, 1977; R o b b i n s and Heywood, 1978). In recent years, a number of attempts have been made to define the changes in general RNA composition as well as alterations in the number of specific genes expressed in muscle cells throughout muscle differentiation. Several conclusions are beginning to emerge, a l t h o u g h c o n s i d e r a b l e disagreement exists among individual reports. Changes in the composition of the nucleic acids synthesized during muscle cell differentiation have been studied by using quantitative R N A - D N A hybridization techniques. This technique derives its usefulness from the facts that RNA in a cell is exactly complementary in base sequence to the D N A that directed its synthesis, and that these complementary sequences will form stable hybrids in solution under the proper ionic conditions. When total RNA is extracted from these cells and h y b r i d i z e d against w h o l e - e m b r y o D N A at saturating levels of RNA, or alternatively when an excess of RNA is hybridized with a radioactive complementary DNA (cDNA) synthesized in the presence of avian myeioblastosis virus (AMV) reverse transcriptase, the proportion of a cell's genome being transcribed at any point during muscle differentiation can be determined (Colbert et al., 1976; Ordahl and Caplan, 1976; Paterson and Bishop, 1977; Thi Man and Cole, 1974). Application of these techniques have shown that the percentage of the total genome actively transcribed in proliferating myogenic cells is approximately twice the percentage of the genome transcribed in fully developed myotubes. Therefore, terminal differentiation of muscle cells in culture is apparently accompanied by a drastic decrease in the total number of different proteins ultimately synthesized in the cytoplasm of these myotubes. To o b t a i n a d d i t i o n a l i n f o r m a t i o n on the decrease in number of different RNA species synthesized as myogenic cells differentiate, Thi Man and Cole (1974) also conducted hybridization studies under conditions where DNA was in excess. Such studies make it possible to distinguish between repeated sequences of DNA (which code for ribosomal RNA) and rare or nonrepeated sequences of DNA (which code for messenger RNA). Because formation of R N A - D N A hybrids

847

follows second order kinetics, repeated D N A sequences form hybrids quicker than rare DNA sequences. Analysis of RNA from muscle cell cultures in different stages of differentiation by hybridization with excess DNA showed that 30% of the RNA in replicating myogenic cells was synthesized from repeated sequences of DNA and that the remaining 70% of the RNA in these cells was synthesized from nonrepeated DNA sequences. On the other hand, only 16% of the RNA synthesized in multinucleated myotubes was synthesized from repetitive DNA sequences, and the remaining 84% of the RNA in these cells was synthesized from single-copy DNA sequences (Thi Man and Cole, 1974). Furthermore, the nonrepetitive RNA is apparently present in three major frequency classes. One of these frequency classes contains a small number of mRNA species tentatively identified as coding for myofibrillar proteins, and each of these mRNA species is present at roughly 15,000 copies per nucleus (Paterson and Bishop, 1977). These results suggest that the proportion of mRNA synthesis increases relative to ribosomal RNA during muscle differentiation and that myofibrillar protein mRNA makes up a large fraction of total mRNA in fully differentiated cells. This suggestion is further supported by the fact that, during differentiation of myogenic cells, ribosomal RNA synthesis per unit of DNA decreases fivefold, but m R N A synthesis per unit of DNA decreases only twofold (Clissold and Cole, 1973; Thi Man and Cole, 1972, 1974). D u r i n g this same p e r i o d of muscle development when RNA synthesis is declining markedly, the stability of many species of RNA, including presumptive MHC mRNA, increases up to fivefold (Buckingham et al., 1974, 1976). The drastic decrease in RNA synthesis and the disappearance of all but repair DNA synthesis during differentiation of myogenic cells are also accompanied by a marked decrease in activity of glucose-6-phosphate dehydrogense (Love et aL, 1969). Glucose-6-phosphate dehydrogenase is the first enzyme in the pentose phosphate pathway and is therefore responsible for initiating synthesis of the ribose and d e o x y r i b o s e p o r t i o n of the ribonucleotides and deoxyribonucleotides. It is evident that the molecular events involved in elaboration of the myogenic phenotype result from differential gene expression, and the preceding discussion has presented some of the major fragments of information dealing with these events. There are, however, several additional theoretical considerations about the mechanism of differential gene expression in muscle cells. It now


848

YOUNG AND ALLEN

seems reasonably certain that the total transcript diversity (i.e., the number of different m R N A species) decreases in muscle cells during differentiation (Caplan and Ordahl, 1978; Ordahl and Caplan, 1976; Paterson and Bishop, 1977; Thi Man and Cole, 1974), although it should be noted that at least two reports suggest that transcript diversity increases during commitment to the myogenic phenotype (Affara et aL, 1977; Colbert et al., 1976). This diminished transcript diversity could occur by two alternate mechanisms, and elucidation of the correct one has important implications with respect to the basic mechanism of attainment of muscle phenotype. The first possibility for attaining diminished transcript diversity would be irreversible inactivation of a relatively large number of genes concomitant with activation of a smaller number of new, previously untranscribed regions of the genome. Evidence supporting this combination of gene suppression and gene activation has been discussed already (Devlin and Emerson, 1978). An a l t e r n a t e mechanism resulting in decreased transcript diversity would be simple suppression of a portion of the genome, with n o expression of previously untranscribed regions (Ordahl and Caplan, 1976; Caplan and Ordahl, 1978). This latter view holds that tissue-specific genes, such as those for muscle actin and myosin, are transcribed to a small extent (or are at least capable of being transcribed under proper environmental conditions) in all precursor cells within the myogenic lineage. Thus, increased myofibrillar MHC mRNA transcription would be quantitative rather than qualitative. Experimental evidence in support of the gene repression model of d e v e l o p m e n t was o b t a i n e d f r o m a d d i t i v e hybridization experiments in which an equimolar mixture of premyogenic and myogenic m R N A was hybridized to [3H]-labeled, nonrepetitive DNA from each cell type. If identical subsets of genes were expressed in the two cell types in question, the eventual extent of hybridization of either the RNA mixture or each individual RNA sample would be the same regardless of the D N A source. If, however, the genes expressed in the two cell types were partially or completely nonoverlapping, the hybridization level of the RNA mixture would be higher than the level for each individual RNA sample. The results of such experiments permit measurement of the extent of overlap between the two RNA populations in question, and Ordahl and Caplan (1976) interpreted their results to mean that few, if any, n e w D N A sequences were transcribed following myogenic commitment. However, it should be noted that the assay method used by Ordahl and Caplan (1976) was insensitive,

and the results do not constitute definitive evidence in support of their conclusion. The differences between these two fundamental developmental mechanisms may have an impact on the basic approach toward elucidating the mechanism regulating myofibrillar protein synthesis in muscle tissue. For example, it is'clear that myofibrillar M H C is synthesized, if at all, at an infinitesimally low rate in P M b and other nonmuscle cells, and it is equally clear that m y o t u b e s synthesize a p p . r o x i m a t e l y 30,000 MHC/nucleus/min (Emerson and Beckner, 1975). The gene suppression, gene activation mechanism would suggest that the myofibrillar MHC gene in differentiating muscle cells becomes available for transcription as a result of other synthetic events. These events might entail synthesis of other regulator molecules and their interaction with the myofibrillar MHC gene, causing preferential initiation of RNA synthesis by RNA polymerase on the myofibrillar MHC gene. Thus, MHC synthesis would increase as a result of qualitative de-repression of a previously untranscribed gene s e q u e n c e . The gene s u p p r e s s i o n view of development would predict that expression of the myofibrillar MHC gene is blocked in nonmuscle cells by the physical presence of a regulator molecule or by other unsuitable environmental conditions. This blockage is proposed to be nearly quantitative, but not to the extent that z e r o molecules of myofibrillar M H C m R N A are transcribed. Myofibrillar MHC gene transcription during muscle differentiation would then be initiated either because the synthesis of the regulator molecule became suppressed and thereby released the myofibrillar MHC gene for more frequent initiation of transcription, or because d e v e l o p m e n t a l l y - a s s o c i a t e d a l t e r a t i o n s in environmental conditions allowed RNA polymerase to preferentially interact with the myofibrillar gene cluster. Phenotypically, either of these mechanisms would result in muscle development; however, the actual biochemical mechanism would be radically different. The key q u e s t i o n in u n d e r s t a n d i n g m u s c l e p r o t e i n synthesis is how do these mechanism operate postnatally to regulate the quantity of proteins that accumulate. Any basic experiments dealing with either the efficiency or efficacy of myofibrillar protein accretion must eventually come to grips with this basic mechanism of gene expression. I n s u m m a r y , it is c l e a r t h a t m u s c l e d i f f e r e n t i a t i o n is a c c o m p a n i e d by a v a s t r e o r g a n i z a t i o n of p a t t e r n s of nucleic acid m e t a b o l i s m . D r a m a t i c d e c r e a s e s in D N A polymerase, RNA polymerase, and glucose-6-


GENE TRANSITIONS IN MUSCLE p h o s p h a t e d e h y d r o g e n a s e activities occur; the amount of m R N A synthesized relative to the amount of ribosomal R N A increases; and stability of newly synthesized R N A increases. CONCLUSION

In this manuscript we have documented the evidence that differentiation of skeletal muscle fibers is accomplished by activation of the genes coding for the unique proteins found in adult skeletal muscle tissue. The nature of the molecular activities on the chromosome that permit R N A polymerase to initiate transcription of certain gene sequences but not others in unknown. Once muscle fiber-specific genes have been activated, however, the products of these genes accumulate in muscle f i b e r s in s u f f i c i e n t q u a n t i t i e s t o s u p p o r t contraction. At some point muscle cells recognize that additional proteins are unnecessary, and all gene programs then operate at a maintenance level so that the rate of protein synthesis equals the rate of protein degradation. In order for the quantity of proteins in muscle cells to change as a result of e x o g e n o u s s t i m u l i (i.e., e x e r c i s e , i n a c t i v i t y , denervation), protein synthesis and degradation rates must be differentially affected. In view of what is known about regulation of most biological processes, it follows that there must be feedback mechanisms that control myofibrillar protein content. In the presence of a stimulus such as additional work load, the chemical products of this work, or possibly the resultant unique intracellular conditions, may somehow interact either with the protein synthetic machinery to accelerate protein synthesis or with the c h r o m a t i n to result in transcription of additional mRNA. The transcriptional and post-transcriptional biochemical processes which g o v e r n transient changes in the quantity of existing tissue-specific proteins are referred to as gene modulation. M a n y factors, including prolonged exercise, inactivity, d e n e r v a t i o n , i n j u r y and the n e u r o m u s c u l a r disorders, modulate the activity of the myofibrillar protein genes in adult skeletal muscle. Elucidation of the biochemical mechanism of myofibrillar protein gene modulation will likely have a positive effect on meat animal production, and more basic investigations into the nature of this mechanism are encouraged. LITERATURE CITED

Abbott, J., J. Schiltz, S. Dienstman and H. Holtzer. 1974. The phenotypic complexity of myogenic clones. Proc. Nat. Acad. Sci. USA 71:1506. Adelstein, R. S. and M. A. Conti. 1975. Phosphorylation

849

of platelet myosin increases actin-aetivated myosin ATPase activity. Nature 256:597. Adelstein, R. S., M. A. Conti and W. Anderson. 1973. Phosphorylation of human platelet myosin. Proc. Nat. Acad. Sci. USA 70:3115. Adelstein, R. S., M. A. Conti, G. S. Johnson, I. Pastan and T. D. Pollard. 1972. Isolation and characterization of myosin from cloned mouse fibroblasts. Proe. Nat. Acad. Sci. USA 69:3693. Affara, N. A., M. Jacquet, H. Jakob, F. Jacob, and F. Gros. 1977. Comparison of polysomal polyadenylated RNA from embryonal carcinoma and committed myogenic and erythropoietic cell lines. Cell 12:509. Allen, R. E., M. H. Stromer, D. E. Goll and R. M, Robson. 1977. Myofibrillar protein accumulation in differentiating skeletal muscle. Fed. Proc. 36:2159a. Bischoff, R. and H. Holtzer. 1969. Mitosis and the process of differentiation of myogenic cells in vitro. J. Cell Biol. 41:188. Bischoff, R. and M. Lowe. 1974. Cell surface components and the interaction of myogenic cells. Exploratory Concepts in Muscular Dystrophy. 2:17. Boland, R., A. Martonosi and T. W. Tillack. 1974. Developmental changes in the composition and function of sarcoplasmic retieulum. J. Biol. Chem. 249:612. Buckingham, M. E., D. Caput, A. Cohen, R. G. Whalen and F. Gros. 1974. The synthesis and stability of cytoplasmic messenger RNA during myoblast differentiation in culture. Proe. Nat. Acad. Sci. USA 71:1466. Buckingham, M. E., A. Cohen and F. Gros. 1976. Cytoplasmic distribution of pulse-labeled poly(A)containing RNA, particularly 26S RNA, during myoblast growth and differentiation. J. Mol. Biol. 103:611. Buckley, P. A. and I. R. Konigsberg. 1974. Myogenic fusion and the d u r a t i o n of the post-mitotic gap (G~). Develop. Biol. 37:193. Buckley, P. A. and I. R. Konigsberg. 1977. Do myoblasts in vivo withdraw from the cell cycle? A reexamination. Proe. Nat. Acad. Sci. USA 74:203. Burridge, K. and D. Bray. 1975. Purification and structural analysis of myosin from brain and other non-muscle tissues. J. Mol. Biol. 99:1. Caplan, A. I. and C. P. Ordahl. 1978. Irreversible gene repression model for control of development. Science 201:120. Chi, J. C., S. A. Fellini and H. Holtzer. 1975a. Differences among myosins synthesized in non-myogenic cells, presumptive myoblasts, and myoblast. Proc. Nat. Acad. Sci. USA 72:4999. Chi, J. C. H., N. Rubinstein, K. Strahs and H. Hottzer. 1975b. Synthesis of myosin heavy and light chains in muscle cultures. J. Cell Biol. 67:523. Clissold, P. and R. J. Cole. 1973. Regulation of ribosomal RNA synthesis during mammalian myogenesis in culture. Exptl. Cell Res. 80:159. Cohen, R., M. Pacifici, N. Rubenstein, J. Biehl and H. Holtzer. 1977. Effect of a tumour promoter on myogenesis. Nature 266:538. Colbert, D. A., K. Edwards and J. R. Coleman. 1976. Studies on the organisation of the chicken genome and its expression during myogenesis in vitro. Differentiation 5:91. Devlin, R. B. and C. P. Emerson. 1978. Coordinate regulation of contractile protein synthesis during


850

YOUNG AND ALLEN

myoblast differentiation. Cell 13:599. Elzinga, M., B, J. Maron and R. S. Adelstein. 1976. Human heart and platelet actins are products of different genes. Science 191:94. Emerson, C. P. and S. K. Beckner. 1975. Activation of myosin synthesis in fusing and mononucleated myoblasts. J. Mol. Biol. 93:431. Fambrough, D. M. 1974. Cellular and developmental biology of acetylcholine receptors in skeletal muscle. In E. DeRobertis and J. Schact (Ed.) Neurochem. of Cholinergic Receptors. Raven Press, New York. Fambrough, D., H. C. Hartzell, J. E. Rash and A. K. Ritcbie. 1974. Receptor properties of developing muscle. Annals of the New York Academy of Sciences. Fambrough, D. M. and J. E. Rash. 1971. Development of acetylcholine sensitivity during myogenesis. Develop. Biol. 26:55. Fellini, S. A. and H. Holtzer. 1976. The localization of skeletal light meromyosin in cells of myogenic cultures. Differentiation 6:71. Fischbach, G. D., M. Nameroff and P. G. Nelson. 1971. Electrical properties of chick skeletal muscle fibers developing in cell culture. J. Cell. Physiol. 78:289. Fluck, R. A. and R. C. Strohman. 1973. Acetylcholinesterase activity in developing skeletal muscle cells in vitro. Develop. Biol. 33:417. Friedel, S. C. and D. D. Johnson. 1977. Neurotrophic influences on acetylcholinesteraseisozymes in cultured skeletal muscle. Exp. Neurol. 57:257. Guguen-Guillouzo, C., M.-F. Szajnert, J. Marie, D. Delain and F. Schapira. 1977. Differentiation in vivo and in vitro of pyruvate kinase isozymes in rat muscle. Biochimie 59:65. Gruenstein, E. and A. Rich. 1975. Non-identity of muscle and non-muscle actins. Biochem. Biophys. Res. Commun. 64:472. Hahn, G., P. King and S. J. Yang. 1971. Quantitative changes in unscheduled DNA synthesis in rat muscle cells after differentiation. Nature New Biol. 230:242. Hartzell, H. C. and D. M. Fambrough. 1973. Acetylcholine receptor production and incorporation into membranes of developing muscle fibers. Develop. Biol. 30:153. Hausehka, S. D. 1968. Clonal aspects of muscle development and the stability of the differentiated state. In H. Ursprung (Ed.) The Stability of the Differentiated State. P. 37-55. Springer, Baltimore, MD. Herrmann, H., S. M. Heywood and A. C. Marchok. 1970. Reconstruction of muscle development as a sequence of macromolecular syntheses. Current Topics Develop. Biol. 5:181. Holland, P. C. and D. H. MacLennan. 1976. Assembly of the sarcoplasmic reticulum. Biosynthesis of the adenosine triphosphatase in rat skeletal muscle cell cultures. J. Biol. Chem. 251:2030. Holtzer, H. 1970. Myogenesis. In O. A. Schjeidee and J. de Vellis. (Ed.) Cell Differentiation. Van Nostrand, New York, p. 476-503. Holtzer, H., J. Croop, S. Dientsman, H. Ishikawa and A. P. Somlyo. 1976. Effects of cytochalasin B and colcemide on myogenic cultures. Proc. Nat. Acad. Sci. USA 72:513. Holtzer, H., J. M. Marshall and H. Finck. 1957. An analysis of myogenesis by the use of fluorescent myosin. J. Cell Biol. 3:705. Holtzer, H., K. Strahs, J. Biehl, A. P. Somlyo and H. Ishikawa. 1975. Thick and thin filaments in post-

mitotic mononucleated myoblasts. Sci. 188:943. Ishikawa, H., R. Bischoff and H. Holtzer. 1968. Mitosis and intermediate-sized filaments in developing skeletal muscle. J. Cell Biol. 38:538. John, H. A., M. Patrinou-Georgoulas and K. W. Jones. 1977. Detection of myosin heavy chain mRNA during my0genesis in tissue culture by in vitro and in situ hybridization. Cell 12:501. Jorgensen, A. O., V. I. Kalnins, E. Zubrzycka, and D. H. MacLennan. 1977. Assembly of the sarcoplasmic reticulum. Localization by immunofluorescence of sarcoplasmic reticulum proteins in differentiating rat skeletal muscle cell cultures. J. Cell Biol. 74:287. Keller, J. M. and M. Nameroff. 1974. Induction of creatine phosphokinase in cultures of chick skeletal myoblasts w i t h o u t c o n c o m i t a n t cell fusion. Differentiation 2:19. Kendrick-Jones, J. 1973. The subunit of a vertebrate smooth muscle myosin. Phil. Trans. Roy. Soc. 265:183. Lazarides, E. 1975. Tropomyosin antibody: the specific localization of tropomyosin in non-muscle cells. J. Cell Biol. 65:549. Lazarides, E. and K. Weber. 1974. Actin antibody: the specific visualization of actin filaments in non-muscle cells. Proc. Nat. Acad. Sci. USA 71:2268. Lough, J. and R. Bischoff. 1977. Differentiation of creatine phosphokinase during myogenesis: Quantitative fractionation of isozymes. Develop. Biol. 57:330. Lough, J. W., M. L. Entman, E. L. Bossen and J. L. Hansen. 1972. Calcium accumulation by isolated sarcoplasmic reticulum of skeletal muscle during development of tissue culture. J. Cell Physiol. 80:431. Love, D. S., F. J. Stoddard and J. A. Grasso. 1969. Endocrine regulation of embryonic muscle development: Hormonal control of DNA accumulation, pentose cycle activity and myoblast proliferation. Develop. Biol. 20:563. Lowey, S. and D. Risby. 1971. Light chains from fast and slow muscle myosins. Nature 234:81. MacLennan, D. H. and P. C. Holland. 1975. Calcium transport in sarcoplasmic reticulum. Ann. Rev. of Biophys. and Bioeng. 4:377. MacLennan, D. H., P. Seeman, G. H. Iles and C. C. Yip. 1971. Membrane formation by the adenosine triphosphatase of sarcoplasmic reticulum. J. Biol. Chem. 246:2702. MacLennan, D. H., P. T. S. Wong. 1971. Isolation of a calcium sequestering protein from sarcoplasmic reticulum. Proc. Nat. Acad. Sci. USA 68:1231. MacLennan, D. H., C. C. Yip, G. H. Iles and P. Seeman. 1972. Isolation of sarcoplasmic reticulum proteins. Cold Spring Harbor Symp. Quant. Biol. 37:469. Mannherz, H. G. and R. S. Goody. 1976. Proteins of contractile systems. Ann. Rev. Biochem. 45:427. Marchok, A. C. 1966. An autoradiographic observation of nucleoside incorporation in developing muscle cells. Exptl. Cell Res. 43:214. Marchok, A. C. and J. A. Wolff. 1968. Studies of muscle development. IV. Some characteristics of RNA polymerase activity in isolated nuclei from development chick muscle. Biochim. Biophys. Acta. 155:378. Martonosi, A., R. Boland and R. A. Halpin. 1972. The biosynthesis of sarcoplasmic reticulum membranes and the mechanism of calcium transport. Cold Spring Harbor Syrup. Quant. Biol. 37:455.


GENE TRANSITIONS IN MUSCLE Meissner, G. 1975. Isolation and characterization of two types of sarcoplasmic reticulum vesicles. Biochem. Biophys. Acta. 389:51. Morgan, M. S. V. Perry and J. Ottaway. 1976. Myosin light-chain phosphatase. Biochem. J. 157:687. Morris. G. E., M. Piper and R. Cole. 1976. Do increases in enzyme activities during muscle differentiation reflect expression of new genes? Nature 263:76. Moss, P. S. and R. C. Strohman. 1976. Myosin synthesis by fusion-arrested chick embryo myoblasts in cell culture. Develop. Biol. 48:431. Nameroff, M. 1974. Phospholipase C and the myogenic cell surface. Exploratory Concepts in Muscular Dystrophy 2:32. Okazaki, K. and H. Holtzer. 1966. Myogenesis: Fusion, myosin synthesis and the mitotic cycle. Proc. Nat. Acad. Sci. USA 56:i484. O'Neill, M. C. and F. E. Stockdale. 1972. A kinetic analysis of myogenesis in vitro. J. Cell Biol. 52:52. O'Neil, M. C. and R. C. Strohman. 1969. Changes in DNA polymerase activity associated with cell fusion in cultures of embryonic muscle. J. Cell Physiol. 73:61. O'Neil, M. C. and R. C. Strohman. 1970. Studies on the decline of deoxyribonucleic acid polymerase activity during embryonic muscle cell fusion in vitro. Biochem. 9:2832. Ordahl, C. P. and A. I. Caplan. 1976. Transcriptional diversity in myogenesis. Develop. Biol. 54:61. Ostlund, R. E., I. Pastan and R. S. Adelstein. 1974. Myosin in cultured fibroblasts. J. Biol. Chem. 249:3903. Ostwald, T. J. and D. H. MacLennan. 1974. Isolation of a high affinity calcium-binding protein from sarcoplasmic reticulum. J. Biol. Chem, 249:974. Paterson, B. M. and J. O. Bishop. 1977. Changes in the mRNA population of chick myoblasts during myogenesis in vitro. Cell 12:751. Paterson, B. and J. Prives. 1973. Appearance of acetylcholine receptor in differentiating cultures of embryonic chick breast muscle. J. Cell Biol. 59:241. Paterson, B. and R. C. Strohman. 1972. Myosin systhesis in cultures of differentiating chicken embryo skeletal muscle. Develop. Biol. 29: ! 13. Patrinou-Georgoulas, M. and H. A. John. 1977. The genes and mRNA coding for the heavy chains of chick embryonic skeletal myosin. Cell 12:491. Pollard, T. D. and R. R. Weihing. 1974. Actin and myosin and cell movement. CRC Crit. Rev. in Biochem. 2:1. Robbins, J. and S. M. Heywood. 1978. Quantification of myosin heavy-chain mRNA during myogenesis. European J. Biochem. 82:601. Rubinstein, N. A., J. C. H. Chi and H. Holtzer. 1974. Actin and myosin in a variety of myogenic and nonmyogenic cells. Biochem. Biophys. Res. Commun. 57:438. Sanger, J. W. 1974. The use ofcytochalasin to distinguish myoblast from fibroblast in cultures of developing chick striated muscle. Proc. Nat. Acad. Sci. USA 71:3621. Sarkar, S. 1972. Stoichiometry and sequential removal of light chains of myosin. Cold Spring Harbor Symp. Quant. Biol. 37:!4. Sarkar, S., F. A. Sreter and J. Gergely. 1971. Light chains of myosin from white, red and cardiac muscles. Proc. Nat. Acad. Sci. USA 68:946. Sarzala, M. G., M. Pilarska, E. Zubrzycka and M.

851

Michalak. 1975. Changes in the structure, composition and function of sarcoplasmic-reticulum membrane during development. European J. Biochem. 57:25. Scordilis, S. B. and R. S. Adelstein. 1977. Myoblast myosin phosphorylation is a prerequisite for actin activation. Nature 268:558. Shainberg, A. and H. Brik. 1978. The appearance of acetylcholine receptors triggered by fusion of myoblasts in vitro. FEBS Lett. 88:327. Shainberg, A., G. Yagil and D. Yaffe. 1969. Control of myogenesis in vitro by Ca 2+ concentration in nutritional medium. Exp. Cell Res. 58:163. Shainberg, A., G. Yagil and D. Yaffe. 1971. Alterations in enzymatic activities during muscle differentiation in vitro. Develop. Biol. 25:1. Shimada, Y. 1971. Electron microscope observations on the fusion of chick myoblasts in vitro. J. Cell Biol. 48:128. Small, J. V. and A. Sobiezek. 1977. Ca-regulation of mammalian smooth muscle actomyosin via a kinasephosphatase-dependent phosphorylation and dephosphorylation of the 20,000-Mr light chain of myosin. European J. Biochem. 76:521. Sobieszek, A. 1977. Ca-linked phosphorylation of a light chain of vertebrate smooth muscle myosin. European J. Biochem. 73:477. Stewart, P. S. and D. H. MacLennan. 1974. Surface particles of sarcoplasmic reticulum membranes: Structural features of the adenosine triphosphatase. J. Biol. Chem. 249:985. Stockdale, F. E. 1970. Changing levels of DNA polymerase activity during development of skeletal muscle in vitro. Develop. Biol, 21:462. Stockdale, F. E. 1971. DNA synthesis in differentiating skeletal muscle cells: Initiation by ultraviolet light. Science 171:1145. Stockdale, F. E. and H. Holtzer. 1961. DNA synthesis and myogenesis. Exptl, Cell Res. 24:508. Stockdale, F., K. Okazaki, M. Nameroff and H. Holtzer. 1964. 5-Bromodeoxyuridine: Effect of myogenesis in vitro. Science 146:533. Storti, R. V., D. M. Coen and A. Rich. 1976. Tissuespecific forms of actin in the developing chick. Cell 8:521. Storti, R. V. and A. Rich. 1976. Chick cytoplasmic actin and muscle actin have different structural genes. Proc. Nat. Acad. Sci. USA 73:2346. Stromer, M. H., D. E. Goll, R. B. Young, R. M. Robson and F. C. Parrish, Jr. 1974. Ultrastructural features of skeletal muscle differentiation and development. J. Anim. Sci. 38:1111. Tarikas, H. and D. Schubert. 1974. Regulation of adenylate kinase and creatine kinase activities in myogenic cells. Proc. Nat. Acad. Sci. USA 71:2377. Taylor, E. W. 1972. Chemistry of muscle contraction. Ann. Rev. Biochem. 41:577. Tennyson, V. M., M. Brzin and L. T. Kremzner. 1973. Acetylcholinesterase activity in the myotube and muscle satellite cell of the fetal rabbit. An electron microscopic-cytochemical and biochemical study. J. Histochem. Cytochem. 21:634. Thi Man, N. and R. J. Cole. 1972. RNA synthesis during mammalian myogenesis in culture. Exp. Cell Res. 73:429. Thi Man, N. and R. J. Cole. 1974. Quantitative changes in chromosomal activity during chicken myogenesis in vitro--a DNA-RNA hybridization study. Exp. Cell Res. 83:328.


852

YOUNG AND ALLEN

Tillack, T. W., R. Boland and A. Martonosi. 1974. The ultrastrueture of developing sarcoplasmic reticulum. J. Biol. Chem. 249:624. Tonomura, Y. and F. Oosawa. 1972. Molecular mechanism of contraction. Ann. Rev. Biophys. Bioeng. 1:159. Turner, D. C., V. Maier and H. Eppenberger. 1974. Creatine kinase and aldolase isozyme transitions in cultures of chick skeletal muscle cells. Develop. Biol. 37:63. Turner, D. C., R. Gmur, M. Siegrist, E. Burckhardt and H. M. Eppenberger. 1976. Differentiation in cultures derived from embryonic chicken muscle. I. Musclespecific enzyme changes before fusion in EGTAsynchronized cultures. Develop. Biol. 48:258. Van der Bosch, J., C. Schudt and D. Pette. 1972. Quantitative investigation on Ca ++-and pH-dependence of muscle cell fusion in vitro. Biochem. Biophys. Res. Commun. 48:326. Vertel, B. M. and D. A. Fischman. 1976. Myosin accumulation in mononucleated cells of chick muscle cultures. Develop. Biol. 48:438. Wahrmann, J. P., F. Gros and D. Luzzati. 1973. Phosphorylase and glycogen synthetase during myoblast differentiation. Biochimie 55:457. Weeds, A. G. 1969. Light chains of myosin. Nature 223:1362. Whalen, R. G., G. S. Butler-Browne and F. Gros. 1976.

Protein synthesis and actin heterogeneity in calf muscle cells in culture. Proc. Nat. Acad. Sci. USA 73:2018. Wilson, B. W.~ M. A. Mettler and R. W. Asmundson. 1969. Acetylcholine esterase and non-specific esterase in developing avian tissues: Distribution and molecular weights of esterase in normal and dystrophic embryos and chicks. J. Exp. Zool. 172:49. Wilson, B. W., P. S. Nieberg, C. R. Walker, T. A. Linkhart and D. M. Fry. 1973. Production and release of acetylcholinesterase by cultured chick embryo muscle. Develop. Biol. 33:285. Yablonka, Z. and D. Yaffe. 1976. Synthesis of polypeptides with the properties of myosin light chains directed by RNA extracted from muscle cultures. Proc. Natl. Acad. Sci. USA 73:4599. Yablonka, Z. and D. Yaffe. 1977. Synthesis of myosin light chains and accumulation of translatable mRNA coding for light chain-like polypeptides in differentiating muscle cultures. Differentiation 8:133. Yaffe, D. and S. Fuchs. 1967. Autoradiographic study of the incorporation of uridine-3H during myogenesis in tissue culture. Develop. Biol. 15:33. Zubrzycka, E. and D. H. MacLennan. 1976. Assembly of the sarcoplasmic reticulum. Biosynthesis of calsequestrin in rat muscle cell cultures. J. Biol. Chem. 251:7733.
