Motoneurons regulate myoblast proliferation and patterning in Drosophila

Motoneurons directly influence the differentiation of muscle fibers, regulating features such as muscle fiber type and receptor development. Less well understood is whether motoneurons direct earlier events, such as the patterning of the musculature. In Drosophila, the denervation of indirect flight muscles results in a diminished myoblast population and smaller or missing muscle fibers. We have examined whether the neuron-dependent control of myoblast number is due to regulation of cell division, motoneuron-dependent apoptosis, or nerve-dependent localization and migration of myoblasts. We found that denervation resulted in a reduced rate of cell division, as revealed by BrDU incorporation. There was no change in the frequency of apoptotic myoblasts following denervation. Using time lapse imaging of GFP-expressing myoblasts in vivo in pupae, we observed that despite denervation, the migration and localization of myoblasts remained unchanged. In addition to reducing myoblast proliferation, denervation also altered the segregation of myoblasts into the de novo arising dorso-ventral muscles (DVMs). To address this effect on muscle patterning, we examined the expression of the founder-cell marker Dumbfounded/Kirre (Duf) in imaginal pioneer cells. We show that there is a strong correspondence between cells that express Dumbfounded/Kirre and the number of DVM fibers, consistent with a role for these cells in establishing adult muscles. In the absence of innervation the Duf-positive cells are no longer detected, and muscle patterning is severely disrupted. Our results support a model where specialized founder cells prefigure the adult muscle fibers under the control of the nervous system.     

Mode of formation of the flight muscles in a butterfly Pieris brassicae

The formation and development of the dorsal longitudinal flight muscles of the butterfly Pieris brassicae L have been studied by electron and light microscopy. These imaginal muscles arise from two symmetrical pairs of mesothoracic larval muscles, which are morphologically indistinguishable from the other wall muscles at the beginning of the 5th larval instar. However, 2 days before the end of this instar an accumulation of myoblasts is observed at the median region of these muscle fibres. The muscle fibres are penetrated by the myoblasts and broken into fragments. Progressive dedifferentiation of the larval fibrillar material in each of the muscle fragments is observed during the first days of the pupal development. The myoblasts within the basal lamina of the original larval muscle fibres remain associated with the muscle fragments. Myoblasts then fuse with the larval muscle fragments, which simultaneously fuse with each other. This results in the formation of rudimentary imaginal muscle fibres. The development of these fibres, particularly myofibrillogenesis, is studied until the emergence of the imago.     

Origin and development of the dorso-ventral flight muscles in Chironomus (Diptera; Nematocera)

The origin and development of the dorso-ventral flight muscles (DVM) was studied by light and electron microscopy in Chironomus (Diptera; Nematocera). Chironomus was chosen because unlike Drosophila, its flight muscles develop during the last larval instar, before the lytic process of metamorphosis. Ten fibrillar DVM were shown to develop from a larval muscle associated with myoblasts. This muscle is connected to the imaginal leg discso that its cavity communicates with the adepithelial cells present in the disc; but no migration of myoblasts seems to take place from the imaginal leg disc towards the larval muscle or vice versa. At the beginning of the last larval instar, the myoblasts were always present together with the nerves in the larval muscle. In addition, large larval muscle cells incorporated to the imaginal discs were observed to border on the area occupied by adepithelial cells, and are probably involved in the formation of 4 other fibrillar DVM with adepithelial cells. Three factors seem to determine the number of DVM fibres: the initial number of larval fibres in the Anlage, the fusions of myoblasts with these larval fibres and the number of motor axons in the Anlage. The extrapolation of these observations to Drosophila, a higher dipteran, is discussed.     

Patterning Muscles Using Organizers: Larval Muscle Templates and Adult Myoblasts Actively Interact to Pattern the Dorsal Longitudinal Flight Muscles of Drosophila

Pattern formation in muscle development is often mediated by special cells called muscle organizers. During metamorphosis in Drosophila, a set of larval muscles function as organizers and provide scaffolding for the development of the dorsal longitudinal flight muscles. These organizers undergo defined morphological changes and dramatically split into templates as adult fibers differentiate during pupation. We have investigated the cellular mechanisms involved in the use of larval fibers as templates. Using molecular markers that label myoblasts and the larval muscles themselves, we show that splitting of the larval muscles is concomitant with invasion by imaginal myoblasts and the onset of differentiation. We show that the Erect wing protein, an early marker of muscle differentiation, is not only expressed in myoblasts just before and after fusion, but also in remnant larval nuclei during muscle differentiation. We also show that interaction between imaginal myoblasts and larval muscles is necessary for transformation of the larval fibers. In the absence of imaginal myoblasts, the earliest steps in metamorphosis, such as the escape of larval muscles from histolysis and changes in their innervation, are normal. However, subsequent events, such as the splitting of these muscles, fail to progress. Finally, we show that in a mutant combination, null for Erect wing function in the mesoderm, the splitting of the larval muscles is aborted. These studies provide a genetic and molecular handle for the understanding of mechanisms underlying the use of muscle organizers in muscle patterning. Since the use of such organizers is a common theme in myogenesis in several organisms, it is likely that many of the processes that we describe are conserved.     

Myoblast diversification and ectodermal signaling in Drosophila.

The flight muscles of Drosophila derive from myoblasts found on the third instar disc. We demonstrate that these myoblasts already show distinctive properties and examine how this diversity is generated. In the late larva, Vestigial and low levels of Cut are expressed in myoblasts that will contribute to the indirect flight muscles. Other myoblasts, which express high levels of Cut but no Vestigial, are required for the formation of the direct flight muscles. Vestigial and Cut expression are stabilized by a mutually repressive feedback loop. Vestigial expression begins in the embryo in a subset of adult myoblasts, and Wingless signaling is required later to maintain this expression. Thus, myoblasts are divided into identifiable populations, consistent with their allocation to different muscles, and ectodermal signals act to maintain these differences.     

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.     

Fusion of myocytes with larval muscle remnants during flight muscle development in the Colorado beetle

The transformation of the slow contracting larval m. obliquus lateralis caudalis II during metamorphosis into the asynchronous indirect flight muscle, m. obliquus lateralis dorsalis, in the Colorado beetle, Leptinotarsa decemlineata, was examined by electron microscopy. Particular attention was paid to the fate of the larval muscle fibres, the origin and behaviour of the myoblasts for flight muscle development and the change of the myofibrillar filament lattice of the larva into that of the adult. In the pre-pupal period, the larval muscles dedifferentiate and fragment. At pupation, the muscle fibres consist of cell fragments containing very few myofibrils. The sarcoplasmic reticulum and the transverse tubular system are greatly reduced. The number of myoblasts developed from satellite cells by mitosis increases considerably. They penetrate the muscle fibre and surround the cell fragments. The new fibres of the flight muscle develop from myocytes fused with the larval fragments. The larval basal lamina, surrounding the cell fragments and myoblasts, is present in pupae up to 1 day old. In pupae about 2.5 days old new myofibrils appear that have the adult filament lattice. The insect muscle transformation and the repair of vertebrate muscle after injury show striking resemblances.     

The development of adult abdominal muscles in Drosophila: myoblasts express twist and are associated with nerves.

During metamorphosis, the adult muscles of the Drosophila abdomen develop from pools of myoblasts that are present in the larva. The adult myoblasts express twist in the third larval instar and the early pupa and are closely associated with nerves. Growing adult nerves and the twist-expressing cells migrate out across the developing abdominal epidermis, and as twist expression declines, the myoblasts begin to synthesize beta 3 tubulin. There follows a process involving cell fusion and segregation into cell groups to form multinucleate muscle precursors. These bipolar precursors migrate at both ends to find their correct attachment points. beta 3 tubulin expression continues at least until 51 h APF by which time the adult muscle pattern has been established.     

Founder myoblasts and fibre number during adult myogenesis in Drosophila

We have examined the mechanisms underlying the setting of myotubes and choice of myotube number in adult Drosophila. We find that the pattern of adult myotubes is prefigured by a pattern of duf-lacZ-expressing myoblasts at appropriate locations. Selective expression of duf-lacZ in single myoblasts emerges from generalized, low-level expression in all adult myoblasts during the third larval instar. The number of founders, thus chosen, corresponds to the number of fibres in a muscle. In contrast to the embryo, the selection of individual adult founder cells during myogenesis does not depend on Notch-mediated lateral inhibition. Our results suggest a general mechanism by which multi-fibre muscles can be patterned.     

Development of the indirect flight muscles of Drosophila.

We have followed the pupal development of the indirect flight muscles (IFMs) of Drosophila melanogaster. At the onset of metamorphosis larval muscles start to histolyze, with the exception of a specific set of thoracic muscles. Myoblasts surround these persisting larval muscles and begin the formation of one group of adult indirect flight muscles, the dorsal longitudinal muscles. We show that the other group of indirect flight muscles, the dorsoventral muscles, develops simultaneously but without the use of larval templates. By morphological criteria and by patterns of specific gene expression, our experiments define events in IFM development.     

Oogenesis-flight syndrome in crickets: age-dependent egg production, flight performance, and biochemical composition of the flight muscles in adult female Gryllus bimaculatus

Age-dependent changes in flight performance, biochemical composition of flight muscles, and fresh mass of the flight muscles and ovaries were analysed in adult female two-spotted crickets, Gryllus bimaculatus. After the final moult the flight muscle mass increased significantly to a maximum at days 2 and 3. On day 2 the highest flight activity was also observed. Between days 2 and 3 the ovary weight started to rapidly increase due to vitellogenic egg growth, which continued at a high rate until day 10. With the onset of ovarial growth, flight performance decreased and the flight muscles started to histolyse. A high correlation between flight muscle mass and the content of protein, lipid, glycogen, and free carbohydrate in the flight muscle indicated that energy-rich substrates from the degrading flight muscles were used to fuel oogenesis, although flight muscle histolysis can provide only a small fraction of the substrates needed for egg production. In general, there was a clear trade-off between egg production and flight ability. Surprisingly, however, some females possessed well-developed ovaries but displayed no signs of flight muscle histolysis. This observation was corroborated by flight experiments which revealed that, although most flying females had small ovaries, some of them carried an appreciable amount of mature eggs, and thus, somehow managed to evade the oogenesis-flight syndrome.     

Origin of myoblasts involved in the formation of the flight muscles of a butterfly (Pieris brassicae)

The larval muscle precursors of the dorsal longitudinal flight muscles of Pieris brassicae were examined by electron microscopy at five developmental stages of the fifth larval instar. At the beginning of this instar, a large neuromuscular area (NMA) was observed along the larval muscle precursors, on the side of the muscle where the nerve comes into contact with it. This area was delimited by the basement membrane of the muscle and by a thicker external basal lamina and contained nerve branches, tracheae, neuromuscular endings and cells with a cytoplasm rich is free ribosomes. Cells similar to the ribosome rich cells of the NMA were located along the motor nerves supplying the larval muscle precursors in a compartment joined to the NMA. From 78 hr after the fourth moult onwards, the ribosome-rich cells increased in number, accumulated inside the NMA and in the space around the nerves. Then they penetrated the muscle fibre via the channels of the transverse system. These cells were the myoblasts that later help to form the flight muscles. As regards their earlier origin, they do not seem to derive from cells formed from larval fibre nuclei, but might be present together with the muscle precursors, and along the nerves from the beginning of larval development. The differences with the Nematoceran Diptera are discussed.     

Transplanted wing and leg imaginal discs in Drosophila melanogaster demonstrate interactions between epidermis and myoblasts in muscle formation

 Adult muscle development in Drosophila is intimately associated with the development of the nervous system and epidermis. During metamorphosis, myoblasts from the wing imaginal disc reach target sites on the developing pupal epidermis and begin the formation of multinucleate myofibres of the dorsal thorax. The paths taken by pupal myoblasts could be specified by the nervous system and/or the epidermis. Using genetically marked donor pupal wing and leg discs transplanted onto pupal hosts, we have generated animals that have ectopic wings or legs and have examined the formation of adult muscle types. We show that thoracic myoblasts migrate over both host and donor epidermis when the transplant site on the host is thoracic. However, when the transplant site is on the abdomen, thoracic myoblasts do not migrate over abdominal epidermis. Our results show that the epidermis plays an important role in determining the migration pattern of myoblasts. Since muscles are multinucleate cells that form by the fusion of myoblasts, one way in which their molecular characteristics could be achieved is by some myoblasts acting as ”founders”. These myoblasts could influence the pattern of gene expression of those nuclei that fuse with them. We have examined, again using disc transplant experiments, if myoblasts on discs have the capacity to express fibre-specific genes as distinct from this property being conferred by other extra-discs myoblasts. Our results demonstrate that disc-associated myoblasts can indeed fuse with each other to express fibre-specific genes. We synthesize the results presented here with those from earlier experiments to suggest a mechanism for muscle patterning in the adult thorax. Received: 8 January 1995 / Accepted in revised form: 22 January 1996