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.     

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.     

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.     

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.     

Regulation of specific developmental fates of larval- and adult-type muscles during metamorphosis of the frog Xenopus

During anuran metamorphosis, larval-type myotubes in both trunk and tail are removed by apoptosis, and only trunk muscles are replaced by newly formed adult-type myotubes. In the present study, we clarified the regulatory mechanisms for specific developmental fates of adult and larval muscles. Two distinct (adult and larval) types of myoblasts were found to exist in the trunk, but no or very few adult myoblasts were found in the tail. Each type of myoblast responded differently to metamorphic trigger, 3,3',5-triiodo-L-thyronine (T(3)) in vitro. T(3)-induced cell death was observed in larval myoblasts but not in adult myoblasts. These results suggest that the fates (life or death) of trunk and tail muscles are determined primarily by the differential distribution of adult myoblasts within the muscles. However, a transplantation study clarified that each larval and adult myoblast was not committed to fuse into particular myotube types, and they could form heterokaryon myotubes in vivo. Cell culture experiments suggested that the following two mechanisms are involved in the specification of myotube fate: (1) Heterokaryon myotubes could escape T(3)-induced death only when the proportion of adult nuclei number was higher than 70% in the myotubes. Apoptosis was not observed in any larval nuclei within the surviving heterokaryon myotubes, suggesting the conversion of larval nuclei fate. (2) Differentiation of adult myoblasts was promoted by the factor(s) released from larval myoblasts in a cell type-specific manner. Taken together, the developmental fate of myotubes is determined by the ratio of nuclei types, and the formation of adult nuclei-rich myotubes was specifically enhanced by larval myoblast factor(s).     

Evolution of the molluscan body plan: the case of the anterior adductor muscle of bivalves

The separated shell plates with the rearranged musculature (adductor muscle) is a novelty for bivalves. Despite its importance in the bivalve bodyplan, the development of the anterior adductor muscle remains unresolved. In this study, we investigate the myogenesis of the bivalve species Septifer virgatus to reveal the developmental origin of the larval muscles in bivalves, focusing on the anterior adductor muscle. We observed that larval retractor muscles are differentiated from the ectomesoderm in bivalves, and that the anterior adductor muscles are derived from primordial larval retractor muscles via segregation of the myoblast during the veliger larval stage. Through the comparative study of myogenesis in bivalves and its related taxa, gastropods, we found that both species possess myoblasts that emerge bilaterally and later meet dorsally. We hypothesize that these myoblasts, which are a major component of the main larval retractor in limpets, are homologous to the anterior adductor muscle in bivalves. These observations imply that the anterior adductor muscle of bivalves evolved as a novel muscle by modifying the attachment sites of an existing muscle.     

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.     

Origin and development of the tergotrochanteral muscle in Chironomus (Diptera: Nematocera)

The origin and the development of the tubular tergo-trochanteral muscle (TTD) was studied by light and electron microscopy in Chironomus (Diptera: Nematocera). Unlike the flight muscles, the TTD was found to develop from myoblasts located around a larval axon, without contribution from a larval muscle. The myoblasts fuse together to form myotubes. Innervation of the TTD arises from the larval axon. The myotubes send out sarcoplasmic extensions towards the axon branches issued from the larval axon. The first differentiated synapses are described. The TTD begins to grow later than the flight muscles. The implications of this developmental lag are discussed.     

Myofiber turnover is used to retrofit frog jaw muscles during metamorphosis

Metamorphic reorganization of the head in anuran amphibians entails abrupt restructuring of the jaw complex as larval feeding structures are transformed into their adult configurations. In this morphometric study, light microscopy wa used to analyze the larval maturation and metamorphic transfiguration of the adductor jaw muscles in the leopard frog (Rana pipiens). Larval jaw muscles, first established during embryogenesis, continue to grow by fiber addition until prometamorphosis, stage XII. Thereafter, fiber number remains stable but additional muscle growth continues by hypertrophy of the individual fibers until metamorphic climax. During metamorphic stages XIX-XXIII, a complete involution of all larval myofibers occurs. Simultaneously, within the same muscle beds, a second wave of myogenesis produces myoblasts which are the precursors of adult jaw myofibers. New muscle fibers continue to be added to these muscles well after the completion of metamorphosis; however, the total duration of the postmetamorphic myogenic period has not been defined. These observations provide clear evidence that the entir population of primary myofibers used in larval oral activity disappears from the adductor muscle beds and is replaced by a second wave of myogenesis commencing during climax. These findings indicate that the adductor jaw muscles are prepared for adult feeding by a complicated cellular process that retrofits existing muscle beds with a completely new complement of myofibers.     

Imaginal pioneers prefigure the formation of adult thoracic muscles in Drosophila melanogaster

In insects, specialized mesodermal cells serve as templates to organize myoblasts into distinct muscle fibers during embryogenesis. In the grasshopper embryo, large mesodermal cells called muscle pioneers extend between the epidermal attachment points of future muscle fibers and serve as foci for myoblast fusion. In the Drosophila embryo, muscle founder cells serve a similar function, organizing large numbers of myoblasts into larval muscles. During the metamorphosis of Drosophila, nearly all larval muscles degenerate and are replaced by a set of de novo adult muscles. The extent to which specialized mesodermal cells homologous to the founders and pioneers of the insect embryo are involved in the development of adult-specific muscles has yet to be established. In the larval thorax, the majority of imaginal myoblasts are associated with the imaginal discs. We report here the identification of a morphologically distinct class of disc-associated myoblasts, which we call imaginal pioneers, that prefigures the formation of at least three adult-specific muscles, the tergal depressor of the trochanter and dorsoventral muscles I and II. Like the muscle pioneers of the grasshopper, the imaginal pioneers attach to the epidermis at sites where the future muscle insertions will arise and erect a scaffold for developing adult muscles. These findings suggest that a prior segregation of imaginal myoblasts into at least two populations, one of which may act as pioneers or founders, must occur during development.     

Innervation regulates the metamorphic fates of larval abdominal muscles in the moth, Manduca sexta

 With the onset of metamorphosis, the abdominal muscles of the moth, Manduca sexta, follow one of three developmental fates: maintenance, respecification, or death. The maintained muscles retain their larval size and morphology throughout adult development. The respecified and dying muscles dedifferentiate, which involves regression, nuclear degeneration, and myofibril breakdown. Nuclei in both dying and respecified muscles also proliferate. The amount of nuclear degeneration is greater in the dying muscle fibers, and the amount of nuclear proliferation is greater in the respecified muscles. Four to ten days after pupation, the sizes of the respecified muscles stabilize while the dying muscles are lost. During regression, a subset of the respecified muscle fibers die. The surviving respecified muscle fibers grow and differentiate during the last half of adult development. In respecified muscles, denervation triggers an increased amount of nuclear degeneration and a decreased amount of nuclear proliferation. As a result, denervated respecified fibers experience increased muscle regression including an increased loss of muscle fibers and sometimes muscle death. Surviving respecified fibers still grow and differentiate yet are only 5 to 12% of the control size. Denervation triggers dedifferentiation in maintained muscles, resulting in fiber loss and occasionally muscle death. The percentage of fibers which dedifferentiate varies between different muscles. Denervation also triggers nuclear proliferation, with the amount of nuclear proliferation correlated with the extent of dedifferentiation of the individual muscle fibers. The dedifferentiated maintained fibers subsequently undergo differentiation in the absence of muscle growth. Received: 10 July 1997 / Accepted: 21 April 1998     

Effect of myonuclear number and mitochondrial fusion on Drosophila indirect flight muscle organization and size

Mechanisms involved in establishing the organization and numbers of fibres in a muscle are not completely understood. During Drosophila indirect flight muscle (IFM) formation, muscle growth is achieved by both incorporating hundreds of nuclei, and hypertrophy. As a result, IFMs provide a good model with which to understand the mechanisms that govern overall muscle organization and growth. We present a detailed analysis of the organization of dorsal longitudinal muscles (DLMs), a subset of the IFMs. We show that each DLM is similar to a vertebrate fascicle and consists of multiple muscle fibres. However, increased fascicle size does not necessarily change the number of constituent fibres, but does increase the number of myofibrils packed within the fibres. We also find that altering the number of myoblasts available for fusion changes DLM fascicle size and fibres are loosely packed with myofibrils. Additionally, we show that knock down of genes required for mitochondrial fusion causes a severe reduction in the size of DLM fascicles and fibres. Our results establish the organization levels of DLMs and highlight the importance of the appropriate number of nuclei and mitochondrial fusion in determining the overall organization, growth and size of DLMs.     

Mass and functional capacity of regenerating muscle is enhanced by myoblast transfer

Morphology and functional capacity of homotopically transplanted extensor digitorum longus muscles (EDL) of adult SCID mice that received 1 × 10⁶ myoblasts [stably transfected to express nuclear localizing β-galactosidase under the control of the myosin light-chain 3F promoter/enhancer] 2 days posttransplantation were evaluated 9 weeks after transplantation, to determine whether the injection of exogenous myoblasts had an effect on muscle regeneration. Regenerated muscles that received exogenous myoblasts were compared to similarly transplanted muscles that received (a) no further treatment, or (b) sham injection of the vehicle (without myoblasts) and to unoperated EDL. Nine weeks after myoblast transfer, myofibers containing donor-derived nuclei could be identified after staining with X-gal solution. Judging from its size and poor functional performance compared to muscles subjected to transplantation only, sham injection provided a secondary trauma to the regenerating muscle from which it failed to fully recover. In comparison to the sham-injected muscle, the myoblast-injected muscles weighed 61% more and had 50% more myofibers and 82% more cross-sectional area occupied by myofibers at the muscles' widest girths. Their absolute twitch and tetanic tensions were threefold and twofold greater, respectively, and their specific twitch and tetanic tensions were 71% and 50% greater, respectively, than those of sham-injected muscles. In many parameters, the regenerating muscle subjected to myoblast transfer equaled or exceeded those of muscles that were transplanted only received only one trauma). Absolute twitch and tetanic tensions were 73% and 65% greater, respectively, and specific twitch tensions of the muscles receiving myoblasts were 50% greater than forces generated by muscles subjected to whole-muscle transplantation only. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 185–198, 1997     

Polyploid tissues in the nematode Caenorhabditis elegans

During larval development, the number of somatic nuclei in C. elegans hermaphrodites increases from 558 to 959 (J. E. Sulston and H. R. Horvitz, Dev. Biol. 56, 110-156, 1977; J. E. Sulston et al., Dev. Biol. 100, 64-119, 1983). At the same time, the animals increase about 60-fold in volume. We have measured the DNA contents of several classes of nuclei by quantitating the fluorescence of Hoescht 33258 stained DNA (D. G. Albertson et al., Dev. Biol. 63, 165-178, 1978). Probably all embryonic nuclei, including those of neurons, muscles, hypodermis, and intestine, are diploid at hatching. Neurons, muscles, and nondividing hypodermal nuclei remain diploid throughout larval development. The DNA content of the intestinal nuclei doubles at the end of each larval stage, reaching 32C by the adult stage. New hypodermal cells, generated by division of seam cells in the larval stages, undergo an additional round of DNA replication before fusing with the major syncytium (hyp7, Sulston et al., 1983). Thus the larval hyp7 syncytium comprises a fixed number of diploid embryonic nuclei plus an increasing number of tetraploid postembryonic nuclei. Some of the endoreduplications that occur in the intestinal and hypodermal lineages of C. elegans may correspond to nuclear or cellular divisions in another nematode Panagrellus redivivus (P. W. Sternberg and H. R. Horvitz, Dev. Biol. 93, 181-205, 1982).     

Sca-1 negatively regulates proliferation and differentiation of muscle cells

Satellite cells are tissue-specific stem cells critical for skeletal muscle growth and regeneration. Upon exposure to appropriate stimuli, satellite cells produce progeny myoblasts. Heterogeneity within a population of myoblasts ensures that a subset of myoblasts readily differentiate to form myotubes, whereas other myoblasts remain undifferentiated and thus available for future muscle growth. The mechanisms that contribute to this heterogeneity in myoblasts are largely unknown. We show that satellite cells are Sca-1(neg) but give rise to myoblasts that are heterogeneous for sca-1 expression. The majority of myoblasts are sca-1(neg), rapidly divide, and are capable of undergoing myogenic differentiation to form myotubes. In contrast, a minority population is sca-1(pos), divides slower, and does not readily form myotubes. Sca-1 expression is not static but rather dynamically modulated by the microenvironment. Gain-of-function and loss-of-function experiments demonstrate that sca-1 has a functional role in regulating proliferation and differentiation of myoblasts. Myofiber size of sca-1 null muscles is altered in an age-dependent manner, with increased size observed in younger mice and decreased size in older mice. These studies reveal a novel system that reversibly modulates the myogenic behavior of myoblasts. These studies provide evidence that, rather than being a fixed property, myoblast heterogeneity can be modulated by the microenvironment.     

Early stages of flight muscle development in the blowfly Lucilia cuprina: A light and electron microscopic study

In Lucilia cuprina the dorsal longitudinal flight muscles of the adult develop from two sets of three larval muscles. About the end of the first day after puparium formation the three degenerating larval fibres on each side of the thorax cleave longitudinally into six. The numerous neighbouring myoblasts, which multiply by mitotic divisions, fuse gradually with the developing fibres, so the myoblast nuclei become incorporated into the sarcoplasm to form the muscle syncytium. The changes in length of the muscle fibres are not uniform. Between 30 to 56 hr after puparium formation the fibres shorten, but after this they rapidly increase in length. The structures of the tendon-like strands and of the anterior extremity of the growing fibres are described.     

A NOVEL PATTERN OF APICAL CELL POLYPLOIDY,SEQUENTIAL POLYPLOIDY REDUCTION AND INTERCELLULAR NUCLEAR TRANSFER IN THE RED ALGA POLYSIPHONIA

Nearly a century ago, Rosenvinge published a now-classic paper reporting nuclear transfer between cells of Polysiphonia during secondary pit connection (SPC) formation. While reinvestigating this phenomenon, we discovered that the uninucleate apical cell, which is the progenitor of all cells in the plant, has many times (ca. 64–128 ×) the level of nuclear DNA characteristic of nuclei of gametes or mature pericentral cells. Via a regular sequence of cell divisions, the polyploid apical cell gives rise to tiers of cells, each composed of a number of pericentral cells which surround a single central cell. A large proportion of the nuclear divisions are not accompanied by DNA replication. Thus, as the number of nuclei within elongating pericentral cells increases, the DNA level of nuclei in these cells “cascades” down to the DNA level expected for the particular life history generation (i.e., gametophyte or tetrasporophyte). In mature pericentral cells, the number of nuclei is proportional to the volume of the cell. The pattern of nuclear division, reduction in ploidy level and the timing of intercellular nuclear transfer via SPC formation is regular and characteristic of a species. Nuclei transferred from one cell to an adjacent cell participate in the further nuclear divisions of the recipient cell. The degree of polyploidy in apical cells may determine the number of cells in a “determinant” branch or even the number of cells in “indeterminant” axes. In addition, the highly polyploid state of the germinating spore and its pattern of development may provide for the rapid initial growth so characteristic of this taxon.