The Him gene inhibits the development of Drosophila flight muscles during metamorphosis

During Drosophila metamorphosis some larval tissues escape the general histolysis and are remodelled to form adult tissues. One example is the dorso-longitudinal muscles (DLMs) of the indirect flight musculature. They are formed by an intriguing process in which residual larval oblique muscles (LOMs) split and fuse with imaginal myoblasts associated with the wing disc. These myoblasts arise in the embryo, but remain undifferentiated throughout embryogenesis and larval life, and thus share characteristics with mammalian satellite cells. However, the mechanisms that maintain the Drosophila myoblasts in an undifferentiated state until needed for LOM remodelling are not understood. Here we show that the Him gene is expressed in these myoblasts, but is undetectable in developing DLM fibres. Consistent with this, we found that Him could inhibit DLM development: it inhibited LOM splitting and resulted in fibre degeneration. We then uncovered a balance between mef2, a positive factor required for proper DLM development, and the inhibitory action of Him. Mef2 suppressed the inhibitory effect of Him on DLM development, while Him could suppress the premature myosin expression induced by mef2 in myoblasts. Furthermore, either decreased Him function or increased mef2 function disrupted DLM development. These findings, together with the co-expression of Him and Mef2 in myoblasts, indicate that Him may antagonise mef2 function during normal DLM development and that Him participates in a balance of signals that controls adult myoblast differentiation and remodelling of these muscle fibres. Lastly, we provide evidence for a link between Notch function and Him and mef2 in this balance.     

A dominant negative form of Rac1 affects myogenesis of adult thoracic muscles in Drosophila

Blocking Rac1 function in precursors of the indirect flight muscle of Drosophila severely disrupts muscle formation. The DLM fibers that develop using larval scaffolds are reduced in number and fiber size, while the DVMs, which develop using founder cells, are mostly absent. These adult muscle phenotypes are in part due to a reduced myoblast pool present at the third larval instar. BrDU labeling studies indicated that this is primarily due to a reduction in proliferation. In addition, DVM myoblasts display altered morphology and are unable to segregate into primordia. This defect precedes the evident block in fusion. We also show that the recently described DVM founder cells can be labeled with 22C10 and beta-3 tubulin, and that they are present under conditions of dominant negative Rac1(N17) expression. Despite the presence of founder cells, DVM fiber formation is rarely observed. Although DLM myoblasts are able to segregate around their larval scaffolds, the pace of fusion is reduced and consequently there is a delay in DLM fiber formation. Thus, in addition to its well-established role in fusion, Rac1 is also involved in the regulation of myoblast proliferation and segregation during adult myogenesis. These are two new roles for Rac1 in Drosophila.     

On the cellular regulation of growth and development in skeletal muscle

1. Fibres of skeletal muscle in different mammalian species vary more in number and in their rates of growth than in their ultimate breadth, and they grow more slowly in cattle and man than in rats and mice. Cells of large mammalian species probably divide comparatively slowly in pre-natal life but do so for longer, and thus they attain greater numbers than do their counterparts in smaller mammals. Such cells include the precursors of muscle, and common mechanisms may therefore limit rates of growth before and after muscles form. If some metabolic processes are slower in mammals destined to be large, corresponding trends in age-related cellular changes which ultimately suppress mitotic activity may cause differences between species in the overall size of muscles and in that of other tissues. This is probably an oversimplification. 2. It is difficult to decide how far the rate of growth and the final diameters of muscle fibres reflect the number of myoblasts which initially fuse into myotubes and the number of myoblasts which are subsequently incorporated into individual fibres. New nuclei are probably added with age along the length of a fibre, but it is uncertain whether they then synthesize ribosomes which produce contractile protein. It seems likely that fibres elongate to different extents by adding myoblasts terminally. 3. There is some evidence that myofibrils grow throughout the depth of a fibre by adding new myofilaments to their surface, but there is none that is convincing to the effect that myofibrils form de novo at a fibre's periphery. Ribosome-like structures distributed in the sarcoplasm between myofibrils have been described, and their numbers decline in comparison with those of the myofibrils during growth. Thus, fibres possibly attain their maximum breadth when the loss of superficial filaments from myofibrils exceeds the capacity of ribosomes to replace them. The evidence is inconclusive as to whether myofitrillar protein is broken down and replaced at rates which vary within a muscle or between muscles differing in physiological properties. Sarcoplasmic proteins appear to be replaced more rapidly than those in myofibrils. It is also speculated that muscle proteins are synthesized and degraded more slowly in species which take longer to develop. 4. Observations, with the microscope suggest that new ribosomes appear in cells which are becoming myoblasts. Whether the ribosomes subsequently break down is not established. The evidence that I-somes occur in muscle is inconclusive, as is that for the existence of messenger RNA and its selective synthesis when muscle is forming in the embryo. 5. A decline in the synthesis of RNA occurs as myotubes appear and contractile protein begins to accumulate. The significance of this phenomenon is not known, and in more mature muscle some RNA also appears to fluctuate in a fashion which is unrelated to rates of controlling protein synthesis. Such RNA may occur at the periphery of fibres or in satellite cells. In some instances it may be formed by cells of the connective tissue and capillaries. There are indications that the growth of muscle does not require the continued transport of new RNA and ribosomes into the body of a fibre. 6. As regards the existence of polyribosomes in muscle and the activity of muscle ribosomes in zlitro, most relevant phenomena can be explained if the ribosomes are aggregated, inter ah, by newly completed protein and if observed variations in activity are some function of the residual amounts of nascent protein which remain on the ribosomes. The morphological appearance of ribosomes in myoblasts is difficult to reconcile with the notion of ribosomes linked by messenger RNA. There is also some rather inconclusive evidence that the sarcoplasm varies in the effectiveness with which it supports protein synthesis by ribosomes. 7. Muscle fibres differ markedly in the number of mitochondria which they exhibit in histological sections and in the rate at which the homogenized fibres catalyse the processes of aerobic respiration which occur in mitochondria. It is uncertain how far such variation is determined by the properties of myoblasts and myotubes, by the nature of subsequent contractile activity and by dilution of the mitochondria as myofibrillar protein accumulates. In part, the tendency of fibres richest in mitochondria to be comparatively small may reflect the diversion of energy sources and oxidizable precursors of protein into energy-generating pathways. However, such fibres perhaps also possess fewer nuclei and fewer functional ribosomes. 8. Within a given animal, variation between fibres in the activity of sarcoplasmic enzymes becomes most pronounced after the myoblast stage. Assuming that these sarcoplasmic proteins are increasing by dissimilar amounts, genes in different fibres are perhaps varying in activity, but this has not been studied. It may be that the intermittent and increasingly forceful contractions of developing fast-phasic fibres simply cause them to accumulate increased amounts of amino acids in the pool from which protein is synthesized, so that a generalized stimulation of protein synthesis follows. Sarcoplasmic protein should then accumulate more than myofibrillar protein relative to starting quantities. This is a consequence of sarcoplasmic protein turning over faster. However, in addition, one must postulate that sarcoplasmic enzymes vary in stability between fibre types. It also remains to assess whether such differences reflect the presence of different molecular forms of each enzyme and whether the latter possess dissimilarities of amino-acid sequence or of molecular configuration. Similar unsolved problems arise regarding the ATPase activity of myosin in developing muscles and its variation between fibres.     

Long-term culture of muscle explants from Sparus aurata

Although there are mammalian myoblast cell lines, no fish myoblast cell line has been developed so far. The aim of this study was to develop a culture system of muscle explants for fish, as explants provide an approximation of the in vivo conditions for cell proliferation and differentiation, and enable a close comparison with events in muscle regenerating in vivo. Here we describe the main features of a long-term in vitro culture system for muscle explants from Sparus aurata fry. At the time of sampling, the original fibres were damaged and subsequently degenerated as shown by the loss of parvalbumin (PV) and presence of apoptotic nuclei. This mechanical damage provoked a myogenic response by activation of myogenic precursor cells. After a few days, new mononucleate cells aligned with the original fibres were seen in the explants, some with proliferating cell nuclear antigen (PCNA-) and Myf-5-positive nuclei, indicating proliferation and their myogenic fate. By 1 week, multinucleate cells with desmin immunoreactivity but PCNA- and Myf5-negative nuclei were present, equivalent to differentiated, postmitotic myotubes. Some of these myotubes were also immunoreactive for PV and insulin-like growth factors (IGFs). By 11 days, many of the myotubes were also immunoreactive for myostatin (MSTN). By 23 days, many of the myotubes had increased in diameter, were packed with myofibrils, and were strongly PV-positive and immunoreactive for MSTN, IGF-I and IGF-I receptor. This study shows that a proliferative process occurs in the explants despite the death of the original muscle fibres, and new muscle fibres expressing growth regulators are formed by regeneration from myogenic precursors present in the explants at the time of sampling.     

Unusual organization of desmin intermediate filaments in muscular dysgenesis and TTX-treated myotubes

Cytoskeletal intermediate filaments were studied in muscular dysgenesis (mdg) and tetrodotoxin-treated inactive mouse embryo muscle cultures during myofibrillogenesis. Both muscular dysgenesis and tetrodotoxin-treated muscles are characterized in vitro by a total lack of contractile activity and an abnormal development of myofibrils. We studied the organization of the microtubule and intermediate filament networks with immunofluorescence, using anti-tubulin, anti-vimentin, and anti-desmin antibodies during normal and mdg/mdg myogenesis in vitro. Mdg/mdg myotubes present a heterogeneous microtubule network with scattered areas of decreased microtubule density. At the myoblast stage, cells expressed both vimentin and desmin. After fusion only desmin expression is revealed. In mutant myotubes the desmin network remains in a diffuse position and does not reorganize itself transversely, as it does during normal myogenesis. The absence of a mature organization of the desmin network in mdg/mdg myotubes is accompanied by a lack of organization of myofibrils. The role of muscle activity in the organization of myofibrils and desmin filaments was tested in two ways: (i) mdg/mdg myotubes were rendered active by coculturing with normal spinal cord cells, and (ii) normal myotubes were treated with tetrodotoxin (TTX) to suppress contractions. Mdg/mdg innervated myotubes showed cross-striated myofibrils, whereas desmin filaments remained diffuse. TTX-treated myotubes possessed disorganized myofibrils and a very unusual pattern of distribution of desmin: intensively stained desmin aggregates were superimposed upon the diffuse network. We conclude, on the basis of these results, that myofibrillar organization does not directly involve intermediate filaments but does need contractile activity.     

Changes in muscle structure associated with somatic growth in Idiosepius pygmaeus, a small tropical cephalopod

Mantle muscle tissue of Idiosepius pygmaeus was examined to describe changes in structure and organization associated with growth. Growth in I. pygmaeus|DD was a function of both an increase in muscle fibre number and fibre size within muscle blocks. Continuous fibre production over the observed life span of I. pygmaeus was indicated by the presence of very small muscle fibres (< 1.0 μm in diameter) in substantial proportions in all sizes of individuals. Muscle blocks became larger as animals increased in size, although new muscle blocks were generated in all sizes of individuals. Mantle muscle fibres had a maximum size of 11 μm. Therefore, for an individual to continue increasing muscle block sizes, new fibres must be produced. This is further evidence of continuous fibre production throughout the size range of I. pygmaeus examined. The relative rates of muscle fibre generation and fibre growth depended on the size of the animal and position along the mantle (anterior, mid or posterior mantle). The predominance of small fibres and blocks at the anterior end of the mantle suggested that this was the primary growth region. Mitochondriapoor and mitochondria-rich muscle fibres from small individuals had much larger mitochondrial cores than muscle fibres from larger animals. Changes in the muscle structure are discussed with respect to the metabolic and energetic requirements of I. pygmaeus , and how these may change with growth.     

Multinucleation during myogenesis of the myotome of Xenopus laevis: a qualitative study

Ultrastructural studies of myogenesis in the myotome of Xenopus laevis reveal that the myotubes developed by stage 33/34 have peripheral myofibrils but are still uninucleate with a single large nucleus. By stage 45, the cytoplasm of the muscle cells is filled with myofibrils and there are many small peripheral nuclei, resulting in multinucleate muscle fibres. With the electron microscope, we have examined myotomes from stages 33/34 to 59 of development and some stages were also investigated by autoradiography. There was no evidence from autoradiographic studies for DNA synthesis in muscle cells, and the increase in the number of myonuclei was accompanied by a decrease in their size. Satellite cells were not seen at the myotube stage but were first seen after the cells had become multinucleate, with many small nuclei close together forming rows. Constrictions were frequently observed in the large single nuclei. It is concluded that division of the myonuclei by amitosis is mainly responsible for the multinucleation that occurs during development of the myotome muscle in Xenopus laevis.     

Mechanism of myofibril growth and proliferation in fish muscle.

The mechanisms of myofibril growth proliferation were investigated in the red and white muscles of fish. In both types of muscle the ratio of lattice filament spacings between the Z disk and M line was found to be greater than that required for perfect transformation of a square into a hexagonal lattice. This mismatch was considered to result in the thin filaments being pulled obliquely instead of at right angles to the Z disk. The angle of pull of the thin filaments was measured in longitudinal sections. The splitting process was found to decrease the degree of pull. Splitting was also observed in transverse sections of the peripheral myofibrils. In both red and white fibres these myofibrils were found to commence splitting when they reached a size of approximately 1-2 mum diameter. Evidence from ultrastructural and autoradiographical studies suggested that growth of the myofibrils within the fibres is centrifugal. The outermost myofibrils appear to be the ones which are being built up and which split. The data indicated that in fish muscle a considerable number of filaments may be added to the daughter regions whilst splitting of the myofibril is still continuing.     

Myotomal myogenesis inBombina variegata L.

Summary InBombina variegata, striated myofibrils first appear in G₂ uninucleated primary myoblasts. Multinucleated muscle fibres form later as a result of the fusion of primary myobasts with secondary myoblasts of mesenchymal origin. The nuclei of the polykaryocytes vary in size and DNA content (nuclear dimorphism). The larger nuclei of the primary myoblasts retain tetraploid quantities of DNA, whereas the smaller nuclei of the secondary myoblasts are diploid. From this we conclude that fusion can take place between cells that are in different phases of the cell cycle (G₁–G₂). Our findings are compared with those on myogenesis in other chordate species and are confronted with the current commonly accepted model of vertebrate muscle differentiation.This work is dedicated to Professor Kazimierz Sembrat on his 55-th anniversary of research workThis research was supported in part by Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland     

The fine structure of pigeon breast muscle

The pectoralis major muscle of the pigeon is composed of two types of muscle fibre. In the Type I fibres, the myofibrils are closely packed and there are few mitochondria. The myofibrils in the Type II fibres are separated by numerous columns of large mitochondria and lipid droplets. The membrane systems of the two types of fibre are similar. The triads occur at the Z-line; the sarcoplasmic reticulum is in the form of large terminal cisternae which are joined by narrow longitudinal tubules to a broad central cisterna. The value of morphological criteria in the classification of muscle fibres is discussed.     

Extraocular muscles in the lamprey, Lampetra fluviatilis L. I. Muscle fibres

Six extraocular muscles of the river lamprey, Lampetra fluviatilis L., were studied with the light and electron microscope. On the basis of morphology and histochemistry three types of muscle fibres were distinguished: thin, thick mitochondria-rich and thick multifibrillar fibres. In the thin fibres, 2.8-22.4 microns in diameter, myofibrils are distributed peripherally and show strong ATPase activity. The mitochondria are located paraxially. In the thick mitochondria-rich fibres, 19.4-31.0 microns in diameter, myofibrils are also located peripherally, whereas the central part of the fibre is densely packed with very numerous mitochondria possessing tubular cristae. Thick multifibrillar fibres, with a diameter similar to that of the former type, contain thin myofibrils scattered over the entire cross-section of the fibre. The activity of myofibrillar ATPase is lower in both types of thick fibres than in the thin ones. The tubules of the T system were observed frequently only in the thick multifibrillar fibres. The extraocular muscles of the lamprey are composed of large quantities of muscle fibres. Thin and thick fibres do not form separate layers, but are more or less uniformly distributed throughout the muscle. Many muscle fibres show structural features suggesting their degeneration.     

Metamorphosis of Wing Motor System in the Silk Moth, Bombyx mori: Origin of Wing Motor Neurons

The origin of the peripheral nerve and motor neurons that innervate the adult mesothoracic dorsal longitudinal muscles (DLMs) was examined in the silk moth, Bombyx mori . The anatomical features of the peripheral nerve and motor neurons were investigated by dissection, electron microscopy, and cobalt back-fill staining at different pupal stages. These studies showed that the peripheral nerve (IIN1c) that innervates the adult DLMs originates from a branch (db branch) of the larval mesothoracic dorsal nerve that innervates the larval DLMs. During metamorphosis the larval nerve shortens or lengthens locally without change in its basic branching pattern, and the db branch moves towards the mesothoracic ganglion to become the IIN1c. All the adult DLM motor neurons are from larval ones. Nine of the 14 larval DLM motor neurons survive during metamorphosis to become adult DLM motor neurons, and 5 disappear in early pupal stages.     

Muscle fibre growth dynamics in diploid and triploid rainbow trout

The effect of triploidy on muscle fibre growth was determined by comparing hyperplasia and hypertrophy of white muscle fibres in all-female, diploid and triploid rainbow trout Oncorhynchus mykiss (100–400 mm total length). Conventional morphometry and protein and DNA concentrations were used to assess muscle fibre hyperplasia and hypertrophy in white muscle samples derived from an anterio-dorsal location. Muscle fibre distributions were significantly different between triploids and diploids in trout <300 mm. The proportion of fibres <20 μm was higher in diploids than in triploids and the proportion of fibres in the 20–40 μm category was higher in triploids than in diploids. This indicates that the hyperplastic fibres of triploids are larger than those of diploids. Larger hyperplastic fibres in triploids are probably due to the combined effect of increased nuclear size in triploids and the relatively high nucleus: cell ratio observed in small muscle fibres. These larger fibres may be less favourable to cellular metabolic exchange because of their smaller surface area to volume ratios, and perhaps account for reduced viability and growth observed in triploids during early life stages. On the other hand, the lack of difference in the distribution of fibres <20 μm between diploids and triploids at larger body size ranges (301–400 mm) imply that triploid trout may have higher rates of new fibre recruitment and growth capacity at these sizes. There was no difference between diploid and triploid trout in the mean size of muscle fibres; however, the number of fibres per unit area was reduced by 10% in triploids. No differences were observed in protein or DNA concentrations in muscle tissues between the two genetic groups. Since triploid nuclei have 1·5 times more DNA than diploid nuclei, this deviation from the expected muscle DNA concentration (1·3–1·4 times more DNA in triploids when the 10% reduction in fibre density is considered) suggests that the number of nuclei per muscle fibre is reduced. In both diploids and triploids, mean fibre size increased with body length while fibre density decreased. Similarly, protein concentration in the muscle tissue increased and DNA concentration declined with increasing body length. Protein/DNA ratio was strongly and positively correlated with fibre size. These results demonstrate that changes in DNA and protein concentrations can be used to assess hyperplasia and hypertrophy in muscle tissues. However, the morphometric procedure provides better insight into muscle fibre growth as it enables the direct visualization and analysis of muscle fibre distribution patterns.     

Structural and functional development of cricket ring muscles

The sizes of the unifunctional dorsal longitudinal (DLM) and bifunctional subalar (SA) metathoracic flight muscles of the cricket Teleogryllus oceanicus increase by more than an order of magnitude between the second instar before the terminal molt and the tenth day of adult life. During the same developmental period isometric twitch duration (onset to 50% relaxation, 25 degrees C) varies little, while muscle mitochondrial content increased by a factor of ten as measured by stereological analysis of electron micrographs and citrate synthase activity (mumoles citrate . min-1 . gm protein-1, 25 degrees C). The wing muscles of adults have abundant sarcoplasmic reticulum (SR), narrow myofibrils, and a high volume density of mitochondria. At two molts from adulthood muscles that will later be used in flight behavior also have narrow myofibrils and abundant SR, but unlike muscles at later stages, nymphal muscles have a low volume density of mitochondria. At the terminal molt muscles have at least as much SR as is seen in muscles at the tenth day of adult life, and the myofibrils are also more narrow at the earlier stage. Since there is significant variation in muscle structure and little change in twitch duration during late development, the efficacy of the SR in releasing and resequestering CA2+ is seemingly lower in muscles at the terminal molt, a time of rapid muscle growth.     

Effect of ecdysterone and juvenoid on the developmental involution of flight muscles in Acheta domestica

Morphogenesis and degeneration of the flight muscles in Acheta domestica was studied. The dorso-longitudinal flight muscles (DLMs) degenerate during the fourth day after adult ecdysis and the dorso-ventral flight muscles (DVMs) on the fifteenth day. In the presence of an intact innervation the degeneration of the DLMs can be retarded for 2 days by the injection of ecdysterone into very young adults. This retardation may also result in hypertrophy of the muscle fibres. The injection of ecdysterone, even in high doses, did not affect the flight muscle remnants. No notable changes have been found in the degeneration of DLMs by ovarectomy. Thus, the degeneration of flight muscles and the development of ovaries appear to be independent processes.The DLMs are homogeneous in fibre pattern in respect to succinic dehydrogenase, an important oxidative enzyme, and to ATPase activity, but the muscle fibres do not show any phosphorylase activity.     

Effects of protein synthesis inhibitors on muscle growth in the puparium of Calliphora vomitoria

Methods are described for measuring the length and the myofibril diameter of the sixth dorsal longitudinal flight muscle of 7-, 8- and 9-day old puparia of C. vomitoria. The muscle fibres are divided up into nuclear deliminated areas and the number of myofibrils in one of these areas is found to be constant. The growth of the myofibrils in one of the nuclear deliminated areas is described. Injections of 20 μg of cycloheximide reduces amino acid incorporation into protein to 3–4% of the normal level in 7- and 8-day-old puparia. Injections of cycloheximide does not stop the elongation of the flight muscle but prevents increases in myofibril diameter. These different responses are discussed.     

Growth and multiplication of white skeletal muscle fibres in carp larvae in relation to somatic growth rate

A morphometric analysis of white axial muscle of common carp Cyprinus carpio was undertaken in order to quantify increase in fibre size, fibre nuclei and fibre number in relation to somatic growth rate during early life. In fast-growing carp larvae fed zooplankton, length and height of fibres from the central part of dorsolateral muscle increased at the same rate (0.75) relative to the total length of the larvae during the first 2 weeks of feeding. During this period, the number of nuclei per fibre increased threefold while the number of nuclei per unit fibre surface remained constant. In fast-growing larvae fed a formulated diet, the total cross-sectional area of one epaxial quadrant of white muscle and the total area of white fibres increased at almost the same rate (3.15; 3.23) relative to larval total length during the first 28 days of exogenous feeding. The total number of white fibres increased faster (2.07) relative to the total length of larvae than the mean area of white fibres (1.16). Hyperplasia accounted for 64% of muscle growth in these larvae. The proportion of fibres with a width < 10 μm decreased from 72% at first feeding to 14% 28 days later, while the proportion of fibres with a width >20 μm which was 0% at first feeding increased up to 34% in the same time. The recruitment of new white fibres seemed to be almost the same in the whole muscle quadrant at first feeding and 18 or 28 days later but, 8 days after first feeding, a transient significant recruitment of new fibres was shown at the apex of the myotome. Comparisons between fast- and slow-growing groups of larvae showed that for a given larval total length: (1) the mean width of central white fibres was higher and the proportion of central fibres with a width <10 μm was lower in slow-growing larvae than in fast-growing ones; (2) the total number of white fibres was lower for a higher total cross-sectional area of white muscle in slow-growing larvae than in fast-growing ones. These results suggest that, in Cyprinus carpio larvae, slow-growing conditions are related to a decreased contribution of hyperplasia to muscle growth.