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.     

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.     

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.     

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.     

Ostia, the inflow tracts of the Drosophila heart, develop from a genetically distinct subset of cardial cells.

The homeobox gene tinman and the nuclear receptor gene seven-up are expressed in mutually exclusive dorsal vessel cells in Drosophila, however, the physiological reason for this distinction is not known. We demonstrate that tin and svp-lacZ expression persists through the larval stage to the adult stage in the same pattern of cells expressing these genes in the embryo. In the larva, six pairs of Svp-expressing cells form muscular ostia, which permit hemolymph to enter the heart for circulation, however, more anterior Svp-expressing cells form the wall of the dorsal vessel. During pupation, the adult heart forms from a chimera of larval and imaginal muscle fibers. The portion of the dorsal vessel containing the larval ostia is histolyzed and the anterior Svp-expressing cells metamorphose into imaginal ostia. This is the first demonstration that the significant molecular diversity of cardial cells identified in the embryonic heart correlates with the formation of physiologically and functionally distinct muscle cells in the animal. Furthermore, our experiments define the cellular changes that occur as the larval heart is remodeled into an imaginal structure in an important model organism.     

The role of the NK-homeobox gene slouch (S59) in somatic muscle patterning.

In the Drosophila embryo, a distinct class of myoblasts, designated as muscle founders, prefigures the mature pattern of somatic body wall muscles. Each founder cell appears to be instrumental in generating a single larval muscle with a defined identity. The NK homeobox gene S59 was the first of a growing number of proposed 'identity genes' that have been found to be expressed in stereotyped patterns in specific subsets of muscle founders and their progenitor cells and are thought to control their developmental fates. In the present study, we describe the effects of gain- and loss-of-function experiments with S59. We find that a null mutation in the gene encoding S59, which we have named slouch (slou), disrupts the development of all muscles that are derived from S59-expressing founder cells. The observed phenotypes upon mutation and ectopic expression of slouch include transformations of founder cell fates, thus confirming that slouch (S59) functions as an identity gene in muscle development. These fate transformations occur between sibling founder cells as well as between neighboring founders that are not lineage-related. In the latter case, we show that slouch (S59) activity is required cell-autonomously to repress the expression of ladybird (lb) homeobox genes, thereby preventing specification along the lb pathway. Together, these findings provide new insights into the regulatory interactions that establish the somatic muscle pattern.     

Founder cells regulate fiber number but not fiber formation during adult myogenesis in Drosophila

During insect myogenesis, myoblasts are organized into a pre-pattern by specialized organizer cells. In the Drosophila embryo, these cells have been termed founder cells and play important roles in specifying muscle identity and in serving as targets for myoblast fusion. A group of adult muscles, the dorsal longitudinal (flight) muscles, DLMs, is patterned by persistent larval scaffolds; the second set, the dorso-ventral muscles, DVMs is patterned by mono-nucleate founder cells (FCs) that are much larger than the surrounding myoblasts. Both types of organizer cells express Dumbfounded, which is known to regulate fusion during embryonic myogenesis. The role of DVM founder cells as well as the DLM scaffolds was tested in genetic ablation studies using the UAS/Gal4 system of targeted transgene expression. In both cases, removal of organizer cells prior to fusion, causes formation of supernumerary fibers, suggesting that cells in the myoblast pool have the capacity to initiate fiber formation, which is normally inhibited by the organizers. In addition to the large DVM FCs, some (smaller) cells in the myoblast pool also express Dumbfounded. We propose that these cells are responsible for seeding supernumerary fibers, when DVM FCs are eliminated prior to fusion. When these cells are also eliminated, myogenesis fails to occur. In the second set of studies, targeted expression of constitutively active Ras^V12 also resulted in the appearance of supernumerary fibers. In this case, the original DVM FCs are present, suggesting alterations in cell fate. Taken together, these data suggest that DVM myoblasts are able to respond to cues other than the original founder cell, to initiate fusion and fiber formation. Thus, the role of the large DVM founder cells is to generate the correct number of fibers, but they are not required for fiber formation itself. We also present evidence that the DVM FCs may arise from the leg imaginal disc.     

The development of adult muscles in Drosophila: ablation of identified muscle precursor cells.

A small subset of mesodermal cells continues to express twist in the late embryo of Drosophila. These cells are the precursors of adult muscles. Each late twist-expressing cell begins to divide early in the second larval instar and division continues throughout the second and third instars, resulting in a small clone of twist-expressing cells at puparium formation. Treatment with a DNA-synthesis inhibitor, hydroxyurea (HU), ablates these cells if applied during S-phase of their replication cycle. We ablated twist-expressing lineages in the larva and demonstrated that this results in the absence of subsets of muscles in the adult abdomen and leg. HU treatment during this larval period has no discernible effect on the adult epidermis or innervation. We conclude that the twist-expressing cells identified in the late embryo are the unique primordia of adult muscles. Each primordium is fated to establish 6-10 adult muscle fibres, defined here as a 'muscle fibre group'. Each primordium has a unique fate and, after ablation, is not replaced by neighbouring cells. This unique fate does not rest with a particular founder cell within the primordium but is specified at the primordium level: ablation of a subset of cells within a muscle primordium does not result in an ablation of the resulting muscle group or in a decrease in the number of fibres within that muscle group, but rather results in a uniform decrease in the number of nuclei/fibres throughout the entire muscle. Thus, the twist-expressing primordia in the abdomen appear to be fated to give rise to a particular muscle group but act as an equivalent precursor pool in the formation of that muscle group. Our results permit the conclusion that specific muscle groups in the adult leg arise from restricted pools of twist-expressing adepithelial cells in the larval imaginal disc in a similar fashion. We conclude that the fate restriction of myoblast pools in early development defines elements of the final adult muscle pattern. The fate restriction of myoblast cells may be a result of genetic determination to form a specified muscle group or, alternatively, reflect the spatial isolation of otherwise equivalent cells to form muscle-specific precursor pools.     

The actin nucleator WASp is required for myoblast fusion during adult Drosophila myogenesis

Myoblast fusion provides a fundamental, conserved mechanism for muscle fiber growth. We demonstrate here that the functional contribution of Wsp, the Drosophila homolog of the conserved actin nucleation-promoting factor (NPF) WASp, is essential for myoblast fusion during the formation of muscles of the adult fly. Disruption of Wsp function results in complete arrest of myoblast fusion in all muscles examined. Wsp activity during adult Drosophila myogenesis is specifically required for muscle cell fusion and is crucial both for the formation of new muscle fibers and for the growth of muscles derived from persistent larval templates. Although Wsp is expressed both in fibers and individual myoblasts, its activity in either one of these cell types is sufficient. SCAR, a second major Arp2/3 NPF, is also required during adult myoblast fusion. Formation of fusion-associated actin 'foci' is dependent on Arp2/3 complex function, but appears to rely on a distinct, unknown nucleator. The comprehensive nature of these requirements identifies Arp2/3-based branched actin polymerization as a universal mechanism underlying myoblast fusion.     

Muscle development in the grasshopper embryo. I. Muscles, nerves, and apodemes in the metathoracic leg

Much is known about the development of nerve pathways in the metathoracic limb bud of the grasshopper embryo. In this series of three papers, we report on the development of muscles in the same embryonic appendage. In a fourth paper (E. E. Ball, R. K. Ho, and C. S. Goodman, 1985, J. Neurosci, in press) we examine the development of specific neuromuscular connections for one of these muscles (coxal muscle 133a). In this first paper, we present an overview of the development of muscles, nerves, and apodemes (tendons). We previously reported on a class of large mesodermal cells, called muscle pioneers (MPs), that arises early in development and appears to act as a scaffold for developing muscles and guidance cue for motoneuron growth cones (R. K. Ho, E. E. Ball, and C. S. Goodman, 1983, Nature (London) 301, 66-69). We have used the I-5 monoclonal antibody (which specifically labels the MPs as well as the nerve pathways), HRP immunocytochemistry, and Normarski optics to visualize muscle, nerve, and apodeme development in the embryonic metathoracic limb bud from 27.5% (before the appearance of the MPs) to 55% (after the muscles have attained their basic adult pattern). Cell fusions, cell migration, and cell death all appear to play important roles in the development of MPs. The patterns of muscle development vary greatly, ranging from (i) single MPs for simple muscles (which in the adult have only one bundle of muscle fibers, e.g., coxal muscle 133a), to (ii) arrays of MPs for complex muscles [which in the adult have many bundles of muscle fibers each with separate sites of insertion, e.g., the extensor tibiae (ETi) and flexor tibiae (FlTi) muscles in the femur].     

Muscle founder cells regulate defasciculation and targeting of motor axons in the Drosophila embryo.

During Drosophila embryogenesis, motor axons leave the central nervous system (CNS) as two separate bundles, the segmental nerve (SN) and intersegmental nerve (ISN). From these, axons separate (defasciculate) progressively in a characteristic pattern, initially as nerve branches and then as individual axons, to innervate target muscles [1] [2]. This pattern of branching resembles the outgrowth and defasciculation of motor axons from the neural tube of vertebrate embryos. The factors that trigger nerve branching are unknown. In vertebrate limbs, the branched innervation may depend on mesodermal cues, in particular on the connective tissues that organise the muscle pattern [3]. In Drosophila, the muscle pattern is organised by specific mesodermal cells, the founder myoblasts, which initiate the development of individual muscles [4][5][6]. Founder myoblasts fuse with neighbouring non-founder myoblasts and entrain these to a specific muscle programme, which also determines their innervation [4] [7]. In the absence of mesoderm, ISN and SN can form, but motor axons fail to defasciculate from these bundles [7]. The cue(s) for nerve branching therefore lie within the mesoderm, most likely in the muscles and/or in the precursor cells of the adult musculature [8]. Here, we show that founder myoblasts are the source of the cue(s) that are required to trigger defasciculation and targeted growth of motor axons. Moreover, we found that a single founder myoblast can trigger the defasciculation of an entire nerve branch. This suggests that the muscle field is structured into sets of muscles, each expressing a common defasciculation cue for a particular nerve branch.     

The longitudinal visceral musculature of Drosophila melanogaster persists through metamorphosis

The larval gut of Drosophila is coated with visceral muscles of mesodermal origin. In the midgut region this musculature comprises circular and longitudinal fibres. The complete visceral musculature is described to be removed during metamorphosis and to be replaced by a newly differentiated imaginal tissue resembling the morphology of the larval musculature. However, progenitors of this imaginal visceral musculature have never been detected prior to differentiation. Here I present results indicating that the longitudinal visceral musculature of the midgut completely persists through metamorphosis. Single cells expressing green fluorescent protein (GFP) as a marker were transplanted at the blastoderm stage. All clones contributing to the longitudinal visceral musculature detected in third instar larvae were recovered after metamorphosis in adult flies. Further evidence for the persistence of the larval visceral musculature was obtained from the P[Gal4] insertion line 5053A. It expresses GAL4 specifically in the longitudinal visceral muscles of the midgut of all developmental stages to the adult fly beginning at the end of embryogenesis. By using GFP as a reporter, it was possible to follow these cells through the entire metamorphosis. Although the muscles undergo dramatic morphological changes including the loss of their contractile system, no evidence for a replacement of the larval visceral musculature by imaginal precursor cells was detected.     

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.     

Cell lineage of the thoracic muscles of drosophila

The thorax of the adult Drosophila contains about 80 muscles, which develop from the mesoderm. A new genetic marker was used to map the cell lineage of the myoblasts that form these muscles. Clones of marked cells were produced by irradiation of embryos and larvae, and these were detected in the adult by histochemical staining. The principal findings are that the muscles of each segment have separate origins, and that each becomes compartmented precisely into a dorsal-lineage and a ventral-lineage set of muscles, each set probably being formed by the adepithelial cells found in one imaginal disc. In contrast with the epidermis, the muscles of each thoracic segment are not subdivided into anterior and posterior compartments, and clones of muscle cells that are homozygous for recessivelethal alleles of engralled develop normally.     

Apterous mediates development of direct flight muscles autonomously and indirect flight muscles through epidermal cues

Two physiologically distinct types of muscles, the direct and indirect flight muscles, develop from myoblasts associated with the Drosophila wing disc. We show that the direct flight muscles are specified by the expression of Apterous, a Lim homeodomain protein, in groups of myoblasts. This suggests a mechanism of cell-fate specification by labelling groups of fusion competent myoblasts, in contrast to mechanisms in the embryo, where muscle cell fate is specified by single founder myoblasts. In addition, Apterous is expressed in the developing adult epidermal muscle attachment sites. Here, it functions to regulate the expression of stripe, a gene that is an important element of early patterning of muscle fibres, from the epidermis. Our results, which may have broad implications, suggest novel mechanisms of muscle patterning in the adult, in contrast to embryonic myogenesis.     

A distinct set of founders and fusion-competent myoblasts make visceral muscles in the Drosophila embryo

The embryonic Drosophila midgut is enclosed by a latticework of longitudinal and circular visceral muscles. We find that these muscles are syncytial. Like the somatic muscles they are generated by the prior segregation of two populations of cells: fusion-competent myoblasts and founder myoblasts specialised to seed the formation of particular muscles. Visceral muscle founders are of two classes: those that seed circular muscles and those that seed longitudinal muscles. These specialisations are revealed in mutant embryos where myoblast fusion fails. In the absence of fusion, founders make mononucleate circular or longitudinal fibres, while their fusion-competent neighbours remain undifferentiated.     

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.     

Possible migration of imaginal myoblasts from adjacent nerve sheath into the developing flight muscle of Chironomus

The emplacement of the first imaginal myoblasts along the larval muscles which are precursors of the dorsal longitudinal flight muscles, has been studied in Chironomus (Diptera, Nematocera), by light and electron microscopy. At the beginning of larval life there are no imaginal myoblasts stored along these muscles. These cells are discerned only at the beginning of the last larval instar. They first appear in the median region of the muscles near the neuromuscular junction. Prior to this, however, there are cells possessing the same cytological characteristics as the imaginal myoblasts inside the sheath of the motor nerves that supply the muscles. These observations suggest that myoblasts could arrive by the nerve sheath. The presence of a thick, continuous basal lamina around the larval muscles seems to exclude all other possibility of access to these muscles. The extension of this hypothesis to the Cyclorrhaphan Diptera is discussed.     

Segmental differentiation processes in embryonic muscle development of the grasshopper

To analyse segmental differentiation processes in muscle development, we studied the embryogenesis of the ventral body wall muscles in thoracic and abdominal segments of the grasshopper Schistocerca gregaria at the identified cell level. We visualized differentiating muscle pioneer and muscle precursor cells by staining with a muscle-specific monoclonal antibody and with rhodamine-coupled phalloidin. Our results show that a similar pattern of serially reiterated early muscle pioneers is initially established in all segments. Subsequently, two major segmental differentiation processes occur. First, segment-specific sets of additional, later differentiating muscle pioneers are generated de novo. Second, segment-specific sets of existing early muscle precursors are eliminated through atrophy and eventual loss. These events have consequences for matching homonomy of muscles and their innervating motoneurons. Taken together, these processes in the embryo, in concert with postembryonic differentiation events, play critical roles in shaping the highly specialized muscular structures of the mature animal.     

Cardiac remodeling in Drosophila arises from changes in actin gene expression and from a contribution of lymph gland-like cells to the heart musculature

Understanding the basis of normal heart remodeling can provide insight into the plasticity of the cardiac state, and into the potential for treating diseased tissue. In Drosophila, the adult heart arises during metamorphosis from a series of events, that include the remodeling of an existing cardiac tube, the elaboration of new inflow tracts, and the addition of a layer of longitudinal muscle fibers. We have identified genes active in all these three processes, and studied their expression in order to characterize in greater detail normal cardiac remodeling. Using a Transglutaminase-lacZ transgenic line, that is expressed in the inflow tracts of the larval and adult heart, we confirm the existence of five inflow tracts in the adult structure. In addition, expression of the Actin87E actin gene is initiated in the remodeling cardiac tube, but not in the longitudinal fibers, and we have identified an Act87E promoter fragment that recapitulates this switch in expression. We also establish that the longitudinal fibers are multinucleated, characterizing these cells as specialized skeletal muscles. Furthermore, we have defined the origin of the longitudinal fibers, as a subset of lymph gland cells associated with the larval dorsal vessel. These studies underline the myriad contributors to the formation of the adult Drosophila heart, and provide new molecular insights into the development of this complex organ.