Relationship of primary and secondary myogenesis to fiber type development in embryonic chick muscle.

The formation of fast and slow myotubes was investigated in embryonic chick muscle during primary and secondary myogenesis by immunocytochemistry for myosin heavy chain and Ca2(+)-ATPase. When antibodies to fast or slow isoforms of these two molecules were used to visualize myotubes in the posterior iliotibialis and iliofibularis muscles, one of the isoforms was observed in all primary and secondary myotubes until very late in development. In the case of myosin, the fast antibody stained virtually all myotubes until after stage 40, when fast myosin expression was lost in the slow myotubes of the iliofibularis. In the case of Ca2(+)-ATPase, the slow antibody also stained all myotubes until after stage 40, when staining was lost in secondary myotubes and in the fast primary myotubes of the posterior iliotibialis and the fast region of the iliofibularis. In contrast, the antibodies against slow muscle myosin heavy chain and fast muscle Ca2(+)-ATPase stained mutually exclusive populations of myotubes at all developmental stages investigated. During primary myogenesis, fast Ca2(+)-ATPase staining was restricted to the primary myotubes of the posterior iliotibialis and the fast region of the iliofibularis, whereas slow myosin heavy chain staining was confined to all of the primary myotubes of the slow region of the iliofibularis. During secondary myogenesis, the fast Ca2(+)-ATPase antibody stained nearly all secondary myotubes, while primaries in the slow region of the iliofibularis remained negative. Thus, in the slow region of the iliofibularis muscle, these two antibodies could be used in combination to distinguish primary and secondary myotubes. EM analysis of staining with the fast Ca2(+)-ATPase antibody confirmed that it recognizes only secondary myotubes in this region. This study establishes that antibodies to slow myosin heavy chain and fast Ca2(+)-ATPase are suitable markers for selective labeling of primary and secondary myotubes in the iliofibularis; these markers are used in the following article to describe and quantify the effects that chronic blockade of neuromuscular activity or denervation has on these populations of myotubes.     

Proteoglycan synthesis by primary chick skeletal muscle during in vitro myogenesis

The proteoglycans synthesized by primary chick skeletal muscle during in vitro myogenesis were compared with those of muscle-specific fibroblasts. Cultures of skeletal muscle cells and muscle fibroblasts were separately labeled using [35S] sulfate as a precursor. The proteoglycans of the cell layer and medium were separately extracted and isolated by ion-exchange chromatography on DEAE-Sephacel followed by gel filtration chromatography on Sepharose CL-2B. Two cell layer-associated proteoglycans synthesized both by skeletal muscle cells and muscle fibroblasts were identified. The first, a high molecular weight proteoglycan, eluted from Sepharose CL-2B with a Kav of 0.07 and contained exclusively chondroitin sulfate chains with an average molecular weight greater than 50,000. The second, a relatively smaller proteoglycan, eluted from Sepharose CL-2B with a Kav of 0.61 and contained primarily heparan sulfate chains with an average molecular weight of 16,000. Two labeled proteoglycans were also found in the medium of both skeletal muscle and muscle fibroblasts. A high molecular weight proteoglycan was found with virtually identical properties to that of the high molecular weight chondroitin sulfate proteoglycan of the cell layer. A second, smaller proteoglycan had a similar monomer size (Kav of 0.63) to the cell layer heparan sulfate proteoglycan, but differed from it in that this molecule contained primarily chondroitin sulfate chains with an average molecular weight of 32,000. Studies on the distribution of these proteoglycans in muscle cells during in vitro myogenesis demonstrated that a parallel increase in the relative amounts of the smaller proteoglycans occurred in both the cell layer and medium compared to the large chondroitin sulfate proteoglycan in each compartment. In contrast, muscle-derived fibroblasts displayed a constant ratio of the small proteoglycans of the cell layer and medium fractions, compared to the larger chondroitin sulfate proteoglycan of the respective fraction as a function of cell density. Our results support the concept that proteoglycan synthesis is under developmental regulation during skeletal myogenesis.     

Neuromuscular transmission to identified primary and secondary myotubes: a reevaluation of polyneuronal innervation patterns in rat embryos.

The early morphogenesis of rat skeletal muscle is a biphasic process involving two sequentially generated populations of myotubes. A small population of primary myotubes appears early and is followed by a much larger population of secondary myotubes which appear progressively over a number of days. All previously published electrophysiological studies of developing muscle have failed to appreciate the relevance of biphasic myotube production. Here we reevaluate the status of early motor innervation, taking into account the wide range of sizes and levels of maturity within the two myotube populations. Evoked end-plate potentials (EPPs) were recorded from fibers of E17-20 rat sternocostalis muscles. Impaled fibers were then marked by ejection of HRP from the recording pipet, enabling ultrastructural identification of fibers from which recordings had been made. The average number of synaptic inputs per fiber increased to a peak at E19, and the rate of rise of the EPPs increased with age. The majority of impaled fibers (76%) were subsequently found to be primary myotubes, even at ages when secondary myotubes formed the majority of fibers in the muscle. Electrophysiological studies during early stages of secondary myotube development therefore sample largely from the more mature primary fibers and probably give the wrong impression of the extent and degree of polyneuronal innervation and of synaptic rearrangement within the muscle. In addition, the results show that very young secondary myotubes are distinguished by EPPs of longer latency, slower rate of rise, and smaller size than those of other types of myotubes. These results suggest that young secondary myotubes are predominantly activated by EPPs which originate in adjoining primary myotubes and propagate electronically to the secondary myotube. We propose a new model of early synaptic rearrangement which accommodates the biphasic nature of muscle development. We also suggest that secondary myotubes do not require direct neural input for the initiation of their development.     

Developmental aspects of the innervation of skeletal muscle fibers in the chick embryo

Summary The M. complexus in the chick, commonly called the hatching muscle, undergoes conspicuous growth during the latter stages of embryonic development. Myogenesis of this muscle was compared to that of M. biceps femoris with regard to development of types of muscle fiber and their innervation. In both muscles fibers are of relatively uniform size and show little growth in diameter between 12 days of development and hatching; fibers develop continuously and display a wide range of diameters at all stages.Initial thickenings on the sarcolemma of fibers where axons are closely approximate were first observed at 10 days of development in both muscles. In both muscles fibers are innervated prior to fibers. Terminal axon networks bridge intercellular spaces and contact fibers in different myogenic clusters, fibers that develop on the surface membrane of fibers exhibit focal thickenings of the membrane and some cell projections that are directed toward axon- fiber contacts. These changes occurred only in fibers of M. complexus.At 14 days of embryogenesis, the processes of synaptogenesis and of myelin formation are less advanced in M. biceps femoris than in M. complexus. At this stage a fibers were observed to be innervated in M. complexus, but not yet in M. biceps femoris. Each fiber was observed to be encircled by several preterminal axons.It is concluded that the earlier development of M. complexus is correlated with an equally early development of nerve-muscle interactions.This work was supported in part by a grant from the Muscular Dystrophy Association of America, Inc.Postdoctoral Fellow of the Muscular Dystrophy Association I would like to thank Professor Dr. H. Tamate for his valuable advise. I am also grateful to Dr. L. Doerr, H. Stokes and Judi K. Lund for their advice and skilled technical assistance     

Collagen synthesis in the muscle of developing chick embryos.

Radioactive protein was prepared from the leg muscle of chick embryos, 11, 14, 16 and 17 days old, each injected with radioactive proline and incubated for 30, 60 or 90 min afterwards. The radioactive protein was incubated with collagenase purified by chromatography on a Sephadex G-100 column. Under this condition, only collagen is digested into products soluble in trichloroacetic acid. The relative rate of collagen synthesis was determined by comparing the amount of radioactivity released into the supernatant fraction and that in the residue, by the method of Diegelmann & Peterkofsky [(1972) Dev. Biol. 28, 443--453]. The results show that the rate of collagen synthesis remains at approx. 10% of the rate of synthesis of other non-collagenous proteins during the development of chick embryonic muscle from 11 to 17 days. This suggests that the synthesis of collagen and that of other proteins are co-ordinately regulated at these stages of development.     

Hypaxial muscle migration during primary myogenesis in Xenopus laevis.

In contrast to many vertebrates, the ventral body wall muscles and limb muscles of Xenopus develop at different times. The ventral body wall forms in the tadpole, while limb (appendicular) muscles form during metamorphosis to the adult frog. In organisms that have been examined thus far, a conserved mechanism has been shown to control migratory muscle precursor specification, migration, and differentiation. Here, we show that the process of ventral body wall formation in Xenopus laevis is similar to hypaxial muscle development in chickens and mice. Cells specified for the migratory lineage display an upregulation of pax3 in the ventro-lateral region of the somite. These pax3-positive cells migrate ventrally, away from the somite, and undergo terminal differentiation with the expression of myf-5, followed by myoD. Several other genes are selectively expressed in the migrating muscle precursor population, including neural cell adhesion molecule (NCAM), Xenopus kit related kinase (Xkrk1), and Xenopus SRY box 5 (sox5). We have also found that muscle precursor migration is highly coordinated with the migration of neural crest-derived melanophores. However, by extirpating neural crest at an early stage and allowing embryos to develop, we determined that muscle precursor migration is not dependent on physical or genetic interaction with melanophores.     

Collagen in developing chick muscle in vivo and in vitro

Because of the known role of collagen in chick skeletal muscle differentiation the collagen synthesized by embryonic chick muscle was studied. The major collagen synthesized by this muscle was found to be type I collagen. In addition, the effectiveness of types I, II, III and IV collagens in promoting myoblast fusion in vitro was compared. These collagens were found to be equally effective as in vitro substrates.     

5'-nucleotidase activity in developing chick pectoral muscle.

The activity of the plasma membrane enzyme 5′-nucleotidase varies dramatically during the embryonic development of chick pectoral muscle. The specific activity is greatest at early stages of differentiation (8-day embryos), falls to a minimum on days 12–14, then rises again in older embryos. In cultured muscle cells obtained from embryonic chick muscle the 5′-nucleotidase activity is essentially absent. Muscle cells grown in the presence of bromodeoxyuridine, an inhibitor of muscle differentiation, contain enhanced levels of 5′-nucleotidase activity. These results indicate that 5′-nucleotidase may be absent in muscle fibers, but present in other cells of muscle tissue.     

Distinct roles for adhesion molecules during innervation of embryonic chick muscle

In vitro studies have suggested that the cell adhesion molecules NCAM and G4/L1 contribute to a variety of events during neural development. We have directly tested the role played by these molecules in the process of initial nerve ingrowth and ramification in the embryonic chick iliofibularis muscle by in ovo injections of specific adhesion-blocking antibodies and analysis of the resultant nerve branching pattern in muscle whole mounts. Antibodies against both molecules produced axonal defasciculation, which resulted in an enhanced transverse projection to the fast region of the muscle. In the case of anti-G4/L1, we also observed a large increase in the number of side branches that form from nerve trunks in the slow region and an enhancement of nerve branching in the fast region. Conversely, anti-NCAM produced a striking decrease in both the number and length of side branches in the slow region, and a reduction in nerve branching in the fast region. A similar reduction of nerve branching was obtained following injection of an endosialidase, which removes sialic acid from NCAM, and which was observed to enhance fiber-fiber apposition, presumably by increasing cell adhesion. Based on their biochemical properties in vitro and their in vivo distribution, both NCAM and G4/L1 are in a position to contribute to axon-axon adhesive interactions, whereas NCAM would be expected to also promote axon-myotube interactions. Our observations in fact indicate that these two adhesion molecules play different but complementary roles during muscle innervation and, specifically, that axon-axon fasciculation is influenced by both NCAM and G4/L1 in an anatomically distinct manner to regulate the overall pattern of nerve branching and that NCAM-mediated axon-myotube interactions are necessary for the attainment of the normal stereotyped pattern of nerve branching in both fast and slow regions of this muscle.     

Respiratory capacity of developing chick red and white skeletal muscle

Oxygen consumption, cytochrome oxidase and succinoxidase activity was measured in samples of leg and breast muscle from chick embryos ranging in age from 11 to 19 days. Respiratory parameters increased significantly in both muscle groups during embryonic life. By the later stages of incubation, leg and breast muscles differed significantly in cytochrome and succinoxidase activity. Oxygen uptake between leg and breast muscles did not differ significantly during later development. The results suggest at least a partial pre-natal differentiation of skeletal muscle in the domestic fowl.     

Quantitative measurement of active polysomes of developing chick muscle

The hatching process in embryos of the toad Xenopus laevis consists of two temporally distinct phases. In phase 1, the embryo escapes sequentially from the two outermost jelly layers, J₃ and J₂, and during phase 2 the embryo hatches from the last remaining jelly coat layer J₁ and the fertilization envelope. Phase 1 hatching appears to be a physical process caused by water inbibition of jelly coat layer J₁ and dynamic changes in the volume enclosed by the fertilization envelope. The combined turgor pressure ruptures jelly coat layers J₃ and J₂. The subsequent phase 2 hatching is a result of both physical and chemical processes. Phase 1 hatching exposes layer J₁ to the medium which, in contrast to jelly layers J₂ and J₃ is partially soluble, and permits its gradual dissolution during Phase 2. The embryo secretes a proteolytic enzyme from the frontal region which partially digests the fertilization envelope; subsequent embryo movement ruptures the weakened envelope and completes the hatching process.     

Spatial analysis of limb bud myogenesis: A proximodistal gradient of muscle colony-forming cells in chick embryo leg buds

The regional distribution of myogenic cells in developing chick leg buds has been investigated using an in vitro clonal assay. Leg buds were embedded in gelatin and sectioned at intervals of 100–300 μm utilizing a vibratome, and cells dissected from prospective myogenic areas were analyzed for their ability to form colonies containing multinucleated myotubes. The results show that muscle colony-forming (MCF) cells from stage 23 ( to 4-day incubation) are exclusively of the early morphological type, and are found in the proximal two-thirds of the bud. Late-type MCF cells are first obtained from the proximal sections of stage 24–25 (4- to day) buds; in succeeding stages (26–29), late MCF cells supercede the early MCF cell type in the proximal regions, and extend into progressively more distal sections in a graded fashion. Results from sequential sections suggest that early and late MCF cells are located within the same muscle groups. The proportion of late MCF cells continues to increase throughout this period, until by stage 31 (7 days) only the most distal myogenic regions (the toe muscle regions) have an appreciable proportion of early MCF cells. Clonal plating efficiencies increase throughout the period of analysis, and by stage 31 precisely dissected myogenic regions yield plating efficiencies as high as 36% with greater than 95% of these colonies differentiating as muscle.