Chicken Anterior Latissimus Dorsi Research Articles

Pax3 expression by satellite cells of avian skeletal muscle spindles

Pax3 protein is usually co‐expressed with Pax7 by embryonic dermomyotome cells which give rise to skeletal muscle fibers. Following myogenesis, it is held that Pax3 expression becomes restricted to a few satellite cells (SCs) of select mature muscles. Intrafusal fibers of muscle spindles are thought to persist in an immature state as, unlike extrafusal fibers, they maintain small diameters, developmental myosins, Myf5 expression and high concentrations of SCs. Thus, we hypothesize that Pax3 expression is maintained in intrafusal fibers. Immunohistochemical methods were used to quantify nuclear numbers in chicken anterior latissimus dorsi muscles excised at 9, 30, 62, and 138 days post‐hatch. SCs were identified by their Pax7 expression. In intrafusal fibers, the percentage of SCs expressing Pax3 decreased from 76% at 9 days to 16% at 138 days post‐hatch and, at each age studied, was higher in intrafusal than extrafusal fibers. In extrafusal fibers, after 14% at 9 days, only 0‐2% of SCs expressed Pax3. Muscle spindles offer a novel model for the study of Pax3 expression in SCs. Funded by the Natural Sciences and Engineering Research Council of Canada.Grant Funding SourceNSERC

Physiological factors influencing the growth of skeletal muscle.

The growth of muscle can be regulated by developmental changes or by alterations in hormone levels or in the rate or amount of work demanded. The mechanisms and structures involved in growth processes can be studied by controlling these factors. The models used are chicken anterior latissimus dorsi (ALD) muscle under the influence of overloading and rabbit tibialis anterior (TA) muscle under the influence of chronic nerve stimulation. Both models involve changes in the isoform of myosin that is expressed. Methods of study include quantitative ultrastructural analysis, immunofluorescence and in situ mRNA hybridization. In overloaded chick ALD fibres polysomes are nonuniformly distributed between the myofibrils and in a peripheral annulus even though subcellular concentrations of the new isoform are not found. In normal rabbit muscle the highest concentration of myosin mRNA detected by in situ hybridization is found in the subsarcolemmal zone. In stimulated TA polysomes are found between myofibrils. It appears that the myosin mRNA accumulates at specific cell locations before translation; then diffusion of isomyosin and rapid exchange into myofibrils follows. Therefore, regulation of growth may be possible at the transcriptional, translational and assembly stages.

Slow-tonic muscle fibers and their potential innervation in the turtle,Pseudemys (Trachemys)scripta elegans

A description is provided of the ratio of slow-tonic vs. slow- and fast-twitch fibers for five muscles in the adult turtle, Pseudemys (Trachemys) scripta elegans. The cross-sectional area of each fiber type and an estimation of the relative (weighted) cross-sectional area occupied by the different fiber types are also provided. Two hindlimb muscles (flexor digitorum longus, FDL; external gastrocnemius, EG) were selected on the basis of their suitability for future motor-unit studies. Three neck muscles (the fourth head of testo-cervicis, TeC4; the fourth head of retrahens capitus collique, RCCQ4; transversalis cervicis, TrC) were chosen for their progressively decreasing oxidative capacity. Serial sections were stained for myosin adenosine triphosphatase (ATPase), NADH-diaphorase, and alpha-glycerophosphate dehydrogenase (alpha-GPDH). Conventional fiber-type classification was then performed using indirect markers for contraction speed and oxidative (aerobic) vs. glycolytic (anaerobic) metabolism: i.e., slow oxidative (SO, including slow-twitch and possibly slow-tonic fibers), fast-twitch, oxidative-glycolytic (FOG), and fast-twitch glycolytic (Fg) fibers. Slow-tonic fibers in the SO class were then revealed by directing the monoclonal antibody, ALD-58 (raised against the slow-tonic fiber myosin heavy chain of chicken anterior latissimus dorsi), to additional muscle cross sections. All five of the tested muscles contained the four fiber types, with the ATPase-stained fibers including both slow-tonic and slow-twitch fibers. The extreme distributions of SO fibers were in the predominately glycolytic TrC vs. the predominately oxidative TeC4 muscle (TrC-SO, 9%; FOG, 20%; Fg, 71% vs. TeC4-SO, 58%: FOG, 16%; Fg, 25%). Across the five muscles, the relative prevalence of slow-tonic fibers (4-47%) paralleled that of the SO fibers (9-58%). TeC4 had the highest prevalence of slow-tonic fibers (47%). The test muscles exhibited varying degrees of regional concentration of each fiber type, with the distribution of slow-tonic fibers paralleling that of the SO fibers. In the five test muscles, fiber cross-sectional area was usually ranked Fg > FOG > SO, and slow-twitch always > slow-tonic. In terms of weighted cross-sectional area, which provides a coarse-grain measure of each fiber type's potential contribution to whole muscle force, all five muscles exhibited a higher Fg and lower SO contribution to cross-sectional area than suggested by their corresponding fiber-type prevalence. This was also the case for the slow-twitch vs. slow-tonic fibers. We conclude that slow-tonic fibers are widespread in turtle muscle. The weighted cross-sectional area evidence suggested, however, that their contribution to force generation is minor except in highly oxidative muscles, with a special functional role, like TeC4. There is discussion of: 1) the relationship between the present results and previous work on homologous neck and hindlimb muscles in other nonmammalian species, and 2) the potential motoneuronal innervation of slow-tonic fibers in turtle hindlimb muscles.

Rapid and reciprocal regulation of tenascin-C and tenascin-Y expression by loading of skeletal muscle.

Tenascin-C and tenascin-Y are two structurally related extracellular matrix glycoproteins that in many tissues show a complementary expression pattern. Tenascin-C and the fibril-associated minor collagen XII are expressed in tissues bearing high tensile stress and are located in normal skeletal muscle, predominantly at the myotendinous junction that links muscle fibers to tendon. In contrast, tenascin-Y is strongly expressed in the endomysium surrounding single myofibers, and in the perimysial sheath around fiber bundles. We previously showed that tenascin-C and collagen XII expression in primary fibroblasts is regulated by changes in tensile stress. Here we have tested the hypothesis that the expression of tenascin-C, tenascin-Y and collagen XII in skeletal muscle connective tissue is differentially modulated by mechanical stress in vivo. Chicken anterior latissimus dorsi muscle (ALD) was mechanically stressed by applying a load to the left wing. Within 36 hours of loading, expression of tenascin-C protein was ectopically induced in the endomysium along the surface of single muscle fibers throughout the ALD, whereas tenascin-Y protein expression was barely affected. Expression of tenascin-C protein stayed elevated after 7 days of loading whereas tenascin-Y protein was reduced. Northern blot analysis revealed that tenascin-C mRNA was induced in ALD within 4 hours of loading while tenascin-Y mRNA was reduced within the same period. In situ hybridization indicated that tenascin-C mRNA induction after 4 hours of loading was uniform throughout the ALD muscle in endomysial fibroblasts. In contrast, the level of tenascin-Y mRNA expression in endomysium appeared reduced within 4 hours of loading. Tenascin-C mRNA and protein induction after 4-10 hours of loading did not correlate with signs of macrophage infiltration. Tenascin-C protein decreased again with removal of the load and nearly disappeared after 5 days. Furthermore, loading was also found to induce expression of collagen XII mRNA and protein, but to a markedly lower level, with slower kinetics and only partial reversibility. The results suggest that mechanical loading directly and reciprocally controls the expression of extracellular matrix proteins of the tenascin family in skeletal muscle.

Ribosomes in the skeletal muscle filament lattice.

The compartmentalization of myosin isoforms within a muscle cell (Gauthier: J. Cell Biol. 110:693-701, 1990) suggests that myosin might be assembled directly into thick filaments at sites where it is synthesized. We therefore examined myofibrils by immunoelectron microscopy to determine whether ribosomes are associated with thick filaments under conditions in which new myosin can be identified. We used the embryonic chick anterior latissimus dorsi (ALD), a slow muscle that is induced, by curare, to synthesize a fast myosin isoform that is not normally present. Myosin was localized in situ, using a gold-labeled monoclonal antibody that recognizes the new isoform. The gold marker, as expected, was localized preferentially to the A band. There was an overall increase of fivefold in the number of gold particles per micron2 of A band in the curare-treated compared to the normal ALD, indicating that the labeled isoform was largely newly formed. There was a corresponding preferential distribution of ribosomes at the A band, especially in the H-band region, and the number of ribosomes per micron2 of A band was nearly twice as high in the curare-treated as in the normal muscle. Ribosomes were located between thick filaments, often aligned in rows. We conclude that ribosomes are located within the filament lattice, and therefore that they are available for local myosin synthesis.

Distribution of the slow/cardiac isoform of skeletal muscle Ca(2+)-ATPase in developing and mature tissues of chickens determined using a monoclonal antibody.

We have established that the monoclonal antibody (MAb) AA21, raised against a crude sarcolemmal fraction prepared from adult chicken anterior latissimus dorsi muscle, recognizes the slow twitch/cardiac isoform of calcium ATPase. This was done using a combination of immunohistochemistry at the light and electron microscopic level, the change in the cell distribution in skeletal muscle during development, the molecular weight of the principal protein recognized in Western transfers, and direct comparison with another MAb of known specificity. The antigen is initially expressed by all myotubes at E10 and with development is gradually lost from all presumptive fast fibers. In addition to its immunoreaction and slow extrafusal skeletal muscle fibers, AA21 displays a highly selective immunoreactivity with a number of other cell types in different tissues. The antibody stains a subset of intrafusal muscle fibers and intestinal and arterial smooth muscle, but not venous smooth muscle. In the nervous system, a subpopulation of neurons is intensely stained, most neurons are faintly stained, and glia are not stained at all.

Myosin expression in hypertrophied fast twitch and slow tonic muscles of normal and dystrophic chickens

Disruption of the development program of myosin gene expression has been reported in chicken muscular dystrophy. In the present report, the relationship between muscular dystrophy and the ability of muscle to respond to an increased work load with a transition in the myosin phenotype has been investigated. Hypertrophy of slow tonic anterior latissimus dorsi (ALD) and fast twitch patagialis (PAT) muscles was induced by overloading for 35 days and myosin expression was analyzed by electrophoresis and immunocytochemistry. Normal and dystrophic chicken ALD muscles have nearly identical proportions of SM-1 and SM-2 isomyosins and both exhibit an age-related repression of the SM-1 isomyosin which is enhanced and accelerated by overloading. Immunocytochemistry with anti-myosin heavy chain (MHC) antibodies demonstrates the appearance of nascent myofibers in overloaded ALD muscles from both normal and dystrophic chickens. A minor fast twitch fiber population is also identified which doubles in number with overloading in normal ALD muscles. There are only half as many fast twitch fibers in control dystrophic ALD muscles and this number does not increase with overloading. In contrast to ALD muscles, the isomyosin profile of normal and dystrophic PAT muscles is quite different. There is significantly more FM-3 and significantly less FM-1 isomyosin in the dystrophic PAT muscle. However, both normal and dystrophic PAT muscles exhibit an overload-induced accumulation of the FM-3 isomyosin. Immunocytochemistry reveals that, unlike the normal PAT muscle, the dystrophic PAT muscle contains a population of myofibers which express slow MHCs. As in the ALD muscle, overload-induced hypertrophy is associated with a repression of the SM-1 MHC in these fibers. Nascent myofiber formation does not occur in either normal or dystrophic overloaded PAT muscles.

Differential regulation of actin and myosin isoenzyme synthesis in functionally overloaded skeletal muscle

Overload hypertrophy of the chicken anterior latissimus dorsi muscle is accompanied by a replacement of one myosin isoenzyme (slow myosin-1, SM1) by another (slow myosin-2, SM2). To investigate the molecular mechanisms by which these changes occur, we measured the fractional synthesis rates (ks) in vivo of individual myosin-heavy-chain isoenzymes, total actin and total protein during the first 72 h of muscle growth. Although the ks of total protein and actin were doubled at 24 h, the ks for SM1 and SM2 were depressed. However, the ks of both isomyosins were nearly tripled by 72 h. Despite the increase in muscle size observed at 72 h, the amount of SM1 was reduced by half, indicating increased degradation of SM1. Results of translation of polyribosomes in vitro paralleled the results obtained in vivo. The proportion of total polyadenylylated mRNA in total RNA was increased at 48 and 72 h, but unchanged at 24 h despite the increase in protein synthesis at 24 h. Nuclease-protection analyses indicate that the level of specific SM1 and SM2 mRNAs change in a reciprocal fashion during overload. We conclude that gene-specific and temporal differences exist in the regulatory mechanisms that control overload-induced muscle growth.

Sensitivity to dicholines of membranes from vertebrate and invertebrate muscles

1. The depolarizing effectiveness of azelainylcholine (AzCh, a 7-C-chain dicholine) is about 10 times higher than that of succinylcholine (SCh, a 2-C-chain dicholine) in skeletal muscles of chick, frog and fish, and in body muscles of the earthworm. 2. In the chicken anterior latissimus dorsi (ALD) muscle, AzCh is about 100 times more effective than SCh. 3. In contrast to that in mammalian muscles, the AzCh-SCh sensitivity difference is not increased by denervation in frog muscles. 4. d-Tubocurarine is equally effective in the ALD and in other chicken muscles; its effectiveness is not decreased by denervation in frog muscles. 5. Cells containing muscarinic acetylcholine receptors are weakly sensitive to dicholines or not at all.

Myosin heavy chain composition of single cells from avian slow skeletal muscle is strongly correlated with velocity of shortening during development

We have determined the myosin heavy chain (MHC) composition (using a sensitive sodium dodecyl sulfate-polyacrylamide gel electrophoresis system) and the maximal velocity of shortening ( V max) of single cells from neonatal and adult chicken anterior latissimus dorsi (ALD) muscles. In addition, the MHC, myosin light chain, and regulatory protein (i.e., troponin and tropomyosin subunits) compositions of bundles of ALD fibers were determined at late embryonic, neonatal, and adult ages. At young ages, there are two MHCs in ALD muscle, SM1 and SM2, with SM1 decreasing in relative amount with increasing age, as shown previously by others. The mean V max of single fibers also decreases from neonatal to adult ages. A strong quantitative correlation is demonstrated between the specific MHC composition and V max among individual cells of the ALD muscle at several ages. Since virtually no changes occur in the regulatory protein and myosin light chain compositions of the ALD muscle between late embryonic and adult ages, it appears that the MHC composition of an individual cell in this muscle is the primary determinant of the maximal shortening velocity. These results are the first to illustrate the functional significance of the developmental transition in myosin heavy chain composition of an avian slow skeletal muscle, consistent with our previous findings on mammalian muscle.

Determination of Fiber Types of Chicken Skeletal Muscles Based on Reaction for Actomyosin, Calcium +2 Magnesium+2-Dependent Adenosine Triphosphatase

Muscle fiber subtypes, determined with the actomyosin Ca +2,Mg +2-adenosine triphosphatase (ATPase) reaction in chicken anterior latissimus dorsi and posterior latissimus dorsi muscles, were demonstrated only after acid or alkaline preincubation followed by a 60-min enzyme incubation. In contrast, subtypes were demonstrated in the sartorius muscle either with or without preincubation. A single-step procedure was therefore possible with this muscle. The results were generally similar to those obtained previously with the mycosin Ca +2 -ATPase procedure. Both methods revealed corresponding muscle fiber subtypes, with the exceptions noted below. The actomyosin Ca +2,Mg +2-ATPase procedure, following preincubation at pH 9.4 and 10.3, resulted in a similar reaction intensity in all fiber types. With the myosin Ca +2-ATPase procedure, the IRA (slow) type in anterior latissimus dorsi and sartorius muscles and the I (slow), IIR (fast oxidative-glycolytic), and IIW (fast glycolytic) types in posterior latissimus dorsi muscle had a higher reaction intensity following preincubation at pH 9.4 than at pH 10.3. Fiber Types IIR and IIW in sartorius muscle were easily distinguished with the actomyosin Ca +2,Mg +2-ATPase procedure.

Depression of mechanical function due to active shortening in the chick anterior latissimus dorsi muscle.

1. The depressive effect of shortening on the mechanical properties of the chick anterior latissimus dorsi muscle was assessed by the isovelocity method during tetanic stimulation where the muscle was subjected to a standard conditioning shortening (7% optimum length at approximately 2% maximum shortening velocity, V0) immediately prior to the isovelocity test. Comparison was made to the mechanical properties of non-conditioned (control) contractions. For any given test isovelocity shortening rate, the observed force was always lower if it was preceded by a conditioning shortening. The percentage difference in isovelocity force between conditioned and control contractions was independent of the test shortening velocity. This suggests that shortening lowered the peak isometric force (F0), but did not affect the shape of the force-velocity relation if normalized to a lower F0. 2. Velocity of unloaded shortening, independently measured by the 'slack' method, was unaffected by the conditioning shortening. 3. The magnitude of the force deficit was diminished if the velocity of the conditioning shortening was increased. 4. Recovery of the force deficit was evaluated by allowing a variable period of isometric force redevelopment between the end of the conditioning shortening and the onset of the test isovelocity shortening. The isovelocity force of the conditioned contraction was less than the corresponding control at all times. Complete recovery was not observed with up to 5 s of additional stimulation. However, recovery was observed if the muscle was allowed to relax briefly. 5. Several possible interpretations of our results were considered. Our results are consistent with the hypothesis that the effect of shortening results from non-uniform sarcomere shortening due to a pre-existing heterogeneity of sarcomere strengths. 6. An Appendix describes: (1) how the force-velocity relation would be affected due to the presence of sarcomere strength heterogeneity, and (2) how a model muscle consisting of a heterogeneous population of sarcomeres would be expected to behave following different types of shortenings.

Nascent muscle fiber appearance in overloaded chicken slow-tonic muscle.

The application of a weight overload to the humerus of chickens induces a hypertrophy of anterior latissimus dorsi (ALD) muscle fibers. This growth is accompanied by a rapid and almost complete replacement of one slow-tonic myosin isoform, SM-1, by another slow-tonic isoform, SM-2. In addition, a population of small fibers appears mainly in extrafascicular spaces and, concurrently, three additional myosin bands are detected by gel electrophoresis. Five antibodies against myosin heavy chain (MHC) isoforms were selected as immunocytochemical probes to determine the cellular location and nature of these myosins. The antibodies react with ventricular, fast skeletal muscle and either SM-1 or SM-2, or both the slow-tonic MHCs. The antifast and antiventricular antibodies react with myosin present in the 10-day embryonic ALD muscle but do not react with myosin in posthatch ALD muscle. The small fibers in overloaded muscle contain a myosin isoform characteristically expressed during the embryonic stage of ALD muscle development and therefore are named nascent myofibers. Some of the nascent myofibers do not react with the antibody to both slow-tonic MHCs, indicating the lack of the normal adult slow-tonic myosins which are expressed in 10-day embryos. In order to explore the origin of the nascent fibers, an electron microscopic study was performed. Stereological analysis of the existing fibers shows a stimulation of numbers and sizes of satellite cells. In addition, the volume occupied by nonmuscle and undifferentiated cells increases dramatically. Myotube formation with incipient myofibrils is seen in extrafascicular spaces. These data suggest that new muscle fiber formation accompanies hypertrophy in overloaded chicken ALD muscle and the process may involve satellite cell migration.

Differentiation of the anterior latissimus dorsi muscle of the chicken examined by anti‐myosin monoclonal antibodies

Two new monoclonal antibodies (McAbs), ALD-180 and ALD-88, produced against the myosin of the slow anterior latissimus dorsi (ALD) muscle of the chicken are described. Their specificity for myosin heavy chain (MHC) was established by radioimmunoassay, immunoautoradiography, and immunofluorescence. They were used in conjunction with McAbs MF-14 and MF-30 (which have been characterized previously to be directed against MHC of the fast skeletal muscle) to examine the developmental changes of the chicken ALD muscle. At the 16-day embryonic, early posthatch, and adult stages the ALD muscle fibers differed in their reaction pattern with the McAbs; at the embryonic stage all fibers reacted strongly with ALD-180 and weakly with ALD-88 and MF-30; at the early posthatch stage there was a checkerboard pattern with many fibers not reacting with any of these three McAbs; and at the adult stage all fibers reacted strongly with ALD-180 and ALD-88 and weakly with MF-30. The MF-14 antibody did not react with ALD muscle at any developmental stage. The mature pattern of immunoreactivity of the ALD muscle fibers with the antibodies was established only after 9 weeks posthatch, and during this 9-week period the immunofluorescence changes were nonsynchronous. Based on immunocytochemical evidence of changes in myosin isoform expression, this study clearly demonstrates a distinctive neonatal (early posthatch) stage in the development of the chicken slow muscle.

Transient appearance of a fast myosin heavy chain epitope in slow-type muscle fibres during stretch hypertrophy of the anterior latissimus dorsi muscle in the adult chicken

Myosin expression during hypertrophy of the chicken anterior latissimus dorsi (ALD) muscle was investigated by immunocytochemical procedures using monoclonal antibodies to the fast and slow isoforms of the myosin heavy chain (myosin HC). Antifast antibody 1F9 bound to the adult fast HC of pectoralis muscle and cross-reacted with the HC found in early developing muscle. Antislow antibody 3D1 bound exclusively to the HC of slow myosin 2 (SM2). Stretch hypertrophy of the ALD was produced by attaching a weight to the wing; there was no evidence for a change in fibre number in the muscle. Between 4 and 6 days of stretch there appeared a dramatic increase in the number of fibres staining with the antifast antibody which reached a peak between 12 and 19 days. By this time between 28 and 52% of the fibres in the stretched ALD stained to varying degrees with the antifast antibody compared with much less than 1% in the unstretched control ALD. Most antifast-stained fibres in the stretched muscle also stained with the antislow antibody; the contralateral control muscle showed mostly antislow staining except for the very small number of strongly antifast-stained fibres. By 50 days in some birds and by 80 days in all birds antifast staining had returned to normal. Analysis of the isomyosin composition of the ALD by native gel electrophoresis did not reveal a significant increase in fast myosin content of the hypertrophied muscle even though immunocytochemical staining may have suggested otherwise.

Selected muscle and nerve extracts contain an activity which stimulates myoblast proliferation and which is distinct from transferrin

Extracts from normal chicken anterior latissimus dorsi and dystrophic pectoralis major muscles and from normal chicken sciatic nerves induce a growth stimulation in chicken and rat myogenic cell cultures. Transferrin is only partially responsible for the observed stimulation since the addition of the extracts to transferrin-saturated cultures induces a further growth response and extracts from which transferrin has been removed by immunoabsorption still retain a substantial portion of their stimulation activity. The active fractions of muscle and nerve extracts display heat, acid, and organic solvent inactivation. Gel filtration of ammonium sulfate fractionated activity from the anterior latissimus dorsi muscle suggests the presence of a growth factor in the molecular weight range of 10,000 to 30,000.

Characterization of a monoclonal antibody to myosin specific for mammalian and human type II muscle fibers

We have characterized a monoclonal antibody (McAb), ALD-19, generated against slow myosin from chicken anterior latissimus dorsi (ALD) muscle for use in studies of human and animal muscle fiber types. This McAb bound selectively to the 200 kDa myosin heavy chain band in immunoblots against chicken, rat and human myosins and showed selective staining of A bands in the myofibrils. The reactivity of ALD-19 with various myosin types was quantitated by radioimmunoassays. Fiber type analysis revealed unexpected specificity of McAb ALD-19 for type II mammalian muscle fibers. This antibody should, therefore be useful for identification and quantification of normal type II fibers in human muscle biopsy specimens.

Heterogeneity of type 1 skeletal muscle fibers revealed by monoclonal antibody to slow myosin.

Monoclonal antibodies (McAbs) were generated against slow myosin from the chicken anterior latissimus dorsi (ALD) muscle and their reactivity was checked against fast (pectoralis), slow (ALD), cardiac (ventricular), and smooth (gizzard) myosins by radioimmunoassay (RIA), immunoautoradiography (immunoblots), and indirect immunofluorescence techniques. In RIAs, the McAb (ALD-58) described in this article reacted specifically with slow myosin, with only a weak cross-reactivity to cardiac myosin. In immunoblots against whole muscle homogenates and purified myosins, it bound selectively to the 200 Kd myosin heavy chain band. The ALD-58 antibody stained the fibers of the ALD muscle uniformly but gave three grades of reactions (strong, weak, and negative), with histochemically identified type 1 fibers of sartorius and gastrocnemius muscles demonstrating the immunological heterogeneity of myosins in type 1 skeletal muscle fibers.

Chick myotendinous antigen. I. A monoclonal antibody as a marker for tendon and muscle morphogenesis.

Extracellular matrix components are likely to be involved in the interaction of muscle with nonmuscle cells during morphogenesis and in adult skeletal muscle. With the aim of identifying relevant molecules, we generated monoclonal antibodies that react with the endomysium, i.e., the extracellular matrix on the surface of single muscle fibers. Antibody M1, which is described here, specifically labeled the endomysium of chick anterior latissimus dorsi muscle (but neither the perimysium nor, with the exception of blood vessels and perineurium, the epimysium ). Endomysium labeling was restricted to proximal and distal portions of muscle fibers near their insertion points to tendon, but absent from medial regions of the muscle. Myotendinous junctions and tendon fascicles were intensely labeled by M1 antibody. In chick embryos, " myotendinous antigen" (as we tentatively call the epitope recognized by M1 antibody) appeared first in the perichondrium of vertebrae and limb cartilage elements, from where it gradually extended to the premuscle masses. Around day 6, tendon primordia were clearly labeled. The other structures labeled by M1 antibody in chick embryos were developing smooth muscle tissues, especially aorta, gizzard, and lung buds. In general, tissues labeled with M1 antibody appeared to be a subset of the ones accumulating fibronectin. In cell cultures, M1 antibody binds to fuzzy, fibrillar material on the substrate and cell surfaces of living fibroblast and myogenic cells, which confirms an extracellular location of the antigenic site. The appearance of myotendinous antigen during limb morphogenesis and its distribution in adult muscle and tendon are compatible with the idea that it might be involved in attaching muscle fibers to tendon fascicles. Its biochemical characterization is described in the accompanying paper ( Chiquet , M., and D. Fambrough , 1984, J. Cell Biol. 98:1937-1946).

Fiber number and size in overloaded chicken anterior latissimus dorsi muscle.

The relative contribution of increases in fiber area and number was evaluated in the chicken anterior latissimus dorsi (ALD) muscle in which enlargement was induced by hanging a weight on one wing. ALD muscles from wings to which weights had been attached for periods ranging from 6 to 65 days weighed an average of 105% (range 22-225%) more than control muscles. Total muscle fiber number, determined by direct counts after nitric acid digestion and fiber dissection, and the frequency of branched fibers were unchanged by muscular enlargement. Fiber cross-sectional area was greater (P less than 0.01) in the enlarged muscles. A close relationship existed (r = 0.78) between actual muscle weight and weight calculated as the product of fiber volume, total fiber number, and muscle density for the control and enlarged muscles. Histochemical staining revealed a conversion of type IIa to type I fibers in the stretched muscles. These results support the concept that skeletal muscle enlargement in response to chronic overload is produced by hypertrophy of preexisting fibers and not be a formation of new fibers.