Dorsal Longitudinal Flight Muscles Research Articles

Beclin-mediated Autophagy Drives Dorsal Longitudinal Flight Muscle Histolysis in the Variable Field Cricket, Gryllus lineaticeps.

Flight muscle histolysis is a widespread strategy used by insects to break down functional flight muscle and modulate the energetic costs associated with flight muscle use and maintenance. The variable field cricket, Gryllus lineaticeps, undergoes histolysis during their transition between dispersal flight and reproduction. Despite the importance of histolysis on insect reproduction and fitness, the molecular mechanisms driving this flight muscle breakdown are not well understood. Here, we show that beclin-mediated autophagy, a conserved lysosomal-dependent degradation process, drives breakdown of dorsal longitudinal flight muscle in female flight-capable G. lineaticeps. We found that female G. lineaticeps activate autophagy in their dorsal longitudinal flight muscle (DLM), but to a greater extent than the neighboring dorsoventral flight muscle (DVM) during histolysis. RNA interference knockdown of beclin, a gene that encodes a critical autophagy initiation protein, delayed DLM histolysis, but did not affect DVM histolysis. This suggests that crickets selectively activate autophagy to break down the DLMs, while maintaining DVM function for other fitness-relevant activities such as walking. Overall, we confirmed that autophagy is a critical pathway used to remodel flight muscle cells during flight muscle histolysis, providing novel insights into the mechanisms underlying a major life history transition between dispersal and reproduction.

Alternative relay regulates the adenosine triphosphatase activity of Locusta migratoria striated muscle myosin.

Locust (Locusta migratoria) has a single striated muscle myosin heavy chain (Mhc) gene, which contains 5 clusters of alternative exclusive exons and 1 differently included penultimate exon. The alternative exons of Mhc gene encode 4 distinct regions in the myosin motor domain, that is, the N-terminal SH3-like domain, one lip of the nucleotide-binding pocket, the relay, and the converter. Here, we investigated the role of the alternative regions on the motor function of locust muscle myosin. Using Sf9-baculovirus protein expression system, we expressed and purified 5 isoforms of the locust muscle myosin heavy meromyosin (HMM), including the major isoform in the thorax dorsal longitudinal flight muscle (FL1) and 4 isoforms expressed in the abdominal intersegmental muscle (AB1 to AB4). Among these 5 HMMs, FL1-HMM displayed the highest level of actin-activated adenosine triphosphatase (ATPase) activity (hereafter referred as ATPase activity). To identify the alternative region(s) responsible for the elevated ATPase activity of FL1-HMM, we produced a number of chimeras of FL1-HMM and AB4-HMM. Substitution with the relay of AB4-HMM (encoded by exon-14c) substantially decreased the ATPase activity of FL1-HMM, and conversely, the relay of FL1-HMM (encoded by exon-14a) enhanced the ATPase activity of AB4-HMM. Mutagenesis showed that the exon-14a-encoded residues Gly474 and Asn509 are responsible for the elevated ATPase activity of FL1-HMM. Those results indicate that the alternative relay encoded by exon-14a/c play a key role in regulating the ATPase activity of FL1-HMM and AB4-HMM.

Identification and Expression Analysis of a cDNA Encoding Cyclophilin A from Gryllus bimaculatus (Orthoptera: Gryllidae)

Cyclophilin A (CypA), a cytosolic binding protein of cyclosporine A, is an immunosuppressive drug. In this study, CypA cDNA was cloned from the two-spotted cricket Gryllus bimaculatus (gCypA). The protein encoded by gCypA comprises 165 amino acids with a molecular mass of 19.23 kDa and an isoelectric point of 9.38 and possesses three N-glycosylation sites and 17 phosphorylation sites. The secondary and tertiary structures of gCypA were identified, and homology analysis revealed that it shares around 73%-81% sequence identities with other CypA proteins. When the researchers analyzed the expression levels of gCypA mRNA in various tissues, they found that the foregut exhibited nearly the same expression level as that of the dorsal longitudinal flight muscle (the control). However, gCypA mRNA expression in the fat body, Malpighian tubes, and midgut was less than half of that in the dorsal longitudinal flight muscle. Under endoplasmic reticulum stress conditions, gCypA mRNA expression was highest in Malpighian tubules (about two times higher than the expression in the control). Under starvation conditions, gCypA mRNA expression increased to three times that of the dorsal longitudinal flight muscle 6 days after starvation. Nonetheless, its expression levels decreased in Malpighian tubules under all starvation conditions. This study provides insights into the physiological role of gCypA in G. bimaculatus.

Systemic changes in cell size throughout the body of Drosophila melanogaster associated with mutations in molecular cell cycle regulators

Along with different life strategies, organisms have evolved dramatic cellular composition differences. Understanding the molecular basis and fitness effects of these differences is key to elucidating the fundamental characteristics of life. TOR/insulin pathways are key regulators of cell size, but whether their activity determines cell size in a systemic or tissue-specific manner awaits exploration. To that end, we measured cells in four tissues in genetically modified Drosophila melanogaster (rictorΔ2 and Mnt1) and corresponding controls. While rictorΔ2 flies lacked the Rictor protein in TOR complex 2, downregulating the functions of this element in TOR/insulin pathways, Mnt1 flies lacked the transcriptional regulator protein Mnt, weakening the suppression of downstream signalling from TOR/insulin pathways. rictorΔ2 flies had smaller epidermal (leg and wing) and ommatidial cells and Mnt1 flies had larger cells in these tissues than the controls. Females had consistently larger cells than males in the three tissue types. In contrast, dorsal longitudinal flight muscle cells (measured only in males) were not altered by mutations. We suggest that mutations in cell cycle control pathways drive the evolution of systemic changes in cell size throughout the body, but additional mechanisms shape the cellular composition of some tissues independent of these mutations.

Flight Muscle and Wing Mechanical Properties are Involved in Flightlessness of the Domestic Silkmoth, Bombyx mori.

Flight loss has occurred in many winged insect taxa. The flightless silkmoth Bombyx mori, is domesticated from the wild silkmoth, Bombyx mandarina, which can fly. In this paper, we studied morphological characteristics attributed to flightlessness in silkmoths. Three domestic flightless B. mori strains and one B. mandarina population were used to compare morphological components of the flight apparatus, including wing characteristics (shape, forewing area, loading, and stiffness), flight muscle (weight, ratio, and microscopic detail) and body mass. Compared with B. mandarina, B. mori strains have a larger body, greater wing loading, more flexible wings and a lower flight muscle ratio. The arrangement in microscopy of dorsal longitudinal flight muscles (DLFMs) of B. mori was irregular. Comparative analysis of the sexes suggests that degeneration of flight muscles and reduction of wing mechanical properties (stiffness) are associated with silkmoth flightlessness. The findings provide important clues for further research of the molecular mechanisms of B. mori flight loss.

The tymbal muscle of cicada has flight muscle-type sarcomeric architecture and protein expression

BackgroundThe structural and biochemical features of the tymbal (sound-producing) muscle of cicadas were studied by X-ray diffraction and immunochemistry, and compared with those of flight muscles from the same species.ResultsThe X-ray diffraction pattern of the tymbal muscle was very similar to that of the dorsal longitudinal flight muscle: In both muscles, the 2,0 equatorial reflection is much more intense than the 1,1, indicating that both muscles have a flight muscle-type myofilament lattice. In rigor, the first myosin/actin layer line reflection was finely lattice-sampled, indicating that the contractile proteins are arranged with a crystalline regularity as in asynchronous flight muscles. In contrast, the diffraction pattern from the tensor muscle, which modulates the sound by stressing the tymbal, did not show signs of such high regularity or flight muscle-type filament lattice. Electrophoretic patterns of myofibrillar proteins were also very similar in the tymbal muscle and flight muscles, but distinct from those from the tensor or leg muscles. The antibody raised against the flight muscle-specific troponin-I isoform reacted with an 80-kDa band from both tymbal and flight muscles, but with none of the bands from the tensor or leg muscles.ConclusionThe close similarities of the structural and biochemical profiles between the tymbal and the flight muscles suggest the possibility that a set of flight muscle-specific proteins is diverted to the tymbal muscle to meet its demand for fast, repetitive contractions.

Three-dimensional structure of axonal mitochondria reflects the age of drosophila.

This study aimed to reconstruct a three-dimensional map of axonal mitochondria using Fiji and Neurolucida software, and to observe directly the morphology and distribution of mitochondria in axons of motor neurons in dorsal longitudinal flight muscles of drosophila aged 5 days and 20 days, using electron microscopy. Results indicated that there was no difference in the total area and volume of mitochondria between 5-day-old drosophila and 20-day-old drosophila in all sections, but the ratio of mitochondrial total areas to axon total areas, as well as mitochondrial density of 20-day-old drosophila, was lower than that of 5-day-old drosophila. The number of mitochondria, whose volume was less than 1 000 000 μm3, and between 1 000 000 μm3 and 10 000 000 μm3, was higher in 20-day-old drosophila than that in 5-day-old drosophila. The number of mitochondria with a volume between 1 000 000 μm3 and 100 000 000 μm3 was apparently higher than those with a volume less than 1 000 000 μm3 or larger than 100 000 000 μm3. In addition, the number of mitochondria with a volume more than 100 000 000 μm3 was small; however, the volume was nearly 70% of the total volume in both 5-day-old and 20-day-old drosophila. In contrast, the number of mitochondria with a volume between 1 000 000 μm3 and 10 000 000 μm3 was large, but the volume was less than 30% of the total volume. These experimental findings suggest that changes in mitochondrial morphology and number in motor neurons from the dorsal longitudinal muscle of drosophila are present during different ages.

Length Dependent Activation in Synchronous Flight Muscle from Manduca Sexta

In most striated muscles, the amount of force generated at a given concentration of activating calcium is greater at long sarcomere length (SL) than at shorter SL, referred to as “length dependent activation” (LDA). LDA is prominent in mammalian cardiac muscle and underlies the so-called Frank-Starling Law of the Heart” allowing cardiac output to be adjusted on a beat-to-beat basis. The molecular basis for LDA is not yet understood. The dorsal longitudinal flight muscle (DLM) of the Hawkmoth Manduca sexta is an emerging model system for structural and functional studies of muscle. It is a synchronous muscle, requiring a neural impulse for every muscle twitch, as in mammalian skeletal and cardiac striated muscle, but it is structurally similar to the more widely studied asynchronous insect flight muscles of Drosophila and Lethocerus. Its force-length curve has been shown to be remarkably similar to mammalian cardiac muscle (Tu & Daniel, J Exp Biol 207: 2455. 2004) indicating that Manduca flight muscle might be a useful model system to elucidate various aspects of cardiac function in comparative studies. The present studies were undertaken to characterize LDA in Manduca flight muscle. Conditions were found that allowed chemical skinning of the muscles while maintaining good structural order as assessed by by light and X-ray diffraction. Force-pCa curves were collected as a function of SL. Dorsally located DLMs (cooler in vivo) were compared to ventrally located DLM's (warmer in vivo). We found that both dorsal and ventral DLMs show length-dependent activation. Our study also showed that ventrally located DLM's are less cooperative (Hill coefficient nH ∼1-1.2) than the dorsally located DLM's (nH ∼1.8–1.9) which may be related to their different functions in vivo. Supported by NSF IOS 1022058 and NIH RR08630.

Steroid hormone regulation of the voltage‐gated, calcium‐activated potassium channel expression in developing muscular and neural systems

A precise organization of gene expression is required for developing neural and muscular systems. Steroid hormones can control the expression of genes that are critical for development. In this study we test the hypothesis that the steroid hormone ecdysone regulates gene expression of the voltage-gated calcium-activated potassium ion channel, Slowpoke or KCNMA1. Late in adult development of the tobacco hawkmoth Manduca sexta, slowpoke (msslo) levels increased contributing to the maturation of the dorsal longitudinal flight muscles (DLMs) and CNS. We show that critical components of ecdysteroid gene regulation were present during upreglation of msslo in late adult DLM and CNS development. Ecdysteroid receptor complex heterodimeric partner proteins, the ecdysteroid receptor (EcR) and ultraspiracle (USP), and the ecdysone-induced early gene, msE75B, were expressed at key developmental time points, suggesting that ecdysteroids direct aspects of gene expression in the DLMs during these late developmental stages. We provide evidence that ecdysteroids suppress msslo transcription in the DLMs; when titers decline msslo transcript levels increase. These results are consistent with msslo being a downstream gene in an ecdysteroid-mediated gene cascade during DLM development. We also show that the ecdysteroids regulate msslo transcript levels in the developing CNS. These results will contribute to our understanding of how the spatiotemporal regulation of slowpoke transcription contributes to tailoring cell excitability to the differing physiological and behavioral demands during development.

Using White Noise to Probe Actomyosin Cycling Kinetics During Shortening and Lengthening in Drosophila Flight Muscle Fibers

Our laboratory routinely measures demembranated muscle fiber mechanics using small amplitude sinusoidal length perturbation analysis and estimates kinetics of actomyosin cycling and myosin attachment time. We have developed a complementary measurement technique using small, random changes in muscle length (white-noise length stimuli) that simultaneously cover a broad frequency spectrum, comparable with the sinusoidal length perturbations. We find the white-noise technique provides a description of the viscoelastic properties that is consistent with sinusoidal analysis measurements, rapidly capturing much of the physiological behavior associated with contracting muscle fibers. The white-noise technique samples a vast range of system behavior in a fraction of the time required to complete sinusoidal analysis, and does not require the linear response underlying sinusoidal analysis methods. Thus, we combined the white-noise stimuli with a linear shortening and lengthening transients to probe cross-bridge cycling behavior during these periods of varied load. Preliminary measurements using demembranated dorsal longitudinal flight muscle fibers from Drosophila melanogaster indicate faster cross-bridge cycling kinetics during lengthening and slower cycling during shortening, compared to isometric contraction. During isometric contraction we estimate a myosin attachment time of 5.0 ms, and 4.6 versus 5.5 ms during the lengthening and shortening transients, respectively. These initial applications of the white-noise system analysis technique show promise for future studies probing molecular processes that underlie complicated length transients associated with normal muscle contraction.

Flight muscle myofibrillogenesis in the pupal stage ofDrosophilaas examined by X-ray microdiffraction and conventional diffraction

In the asynchronous flight muscles of higher insects, the lattice planes of contractile filaments are strictly preserved along the length of each myofibril, making the myofibril a millimetre-long giant single multiprotein crystal. To examine how such highly ordered structures are formed, we recorded X-ray diffraction patterns of the developing flight muscles of Drosophila pupae at various developmental stages. To evaluate the extent of long-range myofilament lattice order, end-on myofibrillar microdiffraction patterns were recorded from isolated quick-frozen dorsal longitudinal flight muscle fibres. In addition, conventional whole-thorax diffraction patterns were recorded from live pupae to assess the extent of development of flight musculature. Weak hexagonal fluctuations of scattering intensity were observed in the end-on patterns as early as approximately 15 h after myoblast fusion, and in the following 30 h, clear hexagonally arranged reflection spots became a common feature. The result suggests that the framework of the giant single-crystal structure is established in an early phase of myofibrillogenesis. Combined with published electron microscopy results, a myofibril in fused asynchronous flight muscle fibres is likely to start as a framework with fixed lattice plane orientations and fixed sarcomere numbers, to which constituent proteins are added afterwards without altering this basic configuration.

Programmed cell death in flight muscle histolysis of the house cricket

We have characterized the process of flight muscle histolysis in the female house cricket, Acheta domesticus, through analysis of alterations of tissue wet weight, total protein content, and percent shortening of the dorsal longitudinal flight muscles (DLMs). Our objectives were to (1) define the normal course of histolysis in the cricket, (2) analyze the effects of juvenile hormone (JH) removal and replacement, (3) determine the effects of cycloheximide treatment, and (4) examine patterns of protein expression during histolysis. Our results suggest that flight muscle histolysis in the house cricket is an example of an active, developmentally regulated cell death program induced by an endocrine signal. Initial declines of total protein in DLMs indicated the JH signal that induced histolysis occurred by Day 2 and that histolysis was essentially complete by Day 3. Significant reductions in tissue weight and percent muscle shortening were observed in DLMs from Day 3 crickets. Cervical ligation of Day 1 crickets prevented histolysis but this inhibition could be reversed by continual topical treatments with methoprene (an active JH analog) although ligation of Day 2 crickets did not prevent histolysis. A requirement for active protein expression was demonstrated by analysis of synthesis block by cycloheximide and short-term incorporation of 35S-methionine. Treatment with cycloheximide prevented histolysis. Autofluorographic imaging of DLM proteins separated by electrophoresis revealed apparent coordinated regulation of protein expression.

Coordination Between the Electrical Activity of Developing Indirect Flight Muscles and the Firing Activity of a Population of Neurosecretory Cells in the Silkmoth, Bombyx mori

The developing indirect flight muscles of pharate moths are characterized by a rhythmic discharge of a long bout of flight-pattern-like muscle potentials in the absence of contractions. The electrical activity of the dorsal longitudinal flight muscles (DLMs) in the silkmoth, Bombyx mori, was discernible as a cluster of many series of muscle potentials that last for several minutes on day 4 of the pupal period. The duration of the active phases and the period of rhythmic activity gradually increased to a peak value on day 7 or 8 and then declined until the end of the pupal period. Mean duration of the active phases (+/-SD) and the mean period of the rhythmic activity (+/-SD) at the peak were 38.7+/-8.7 min and 74.5+/-7.3 min, respectively. The rhythmic electrical activity of immature DLMs was closely coordinated with the rhythmic (bursting) activity of a population of neurosecretory cells that are known to produce pheromone-biosynthesis activating neuropeptide (PBAN) and its related peptides, which belong to the multifunctional peptide family, pyrokinin/PBAN. The DLMs always became active a few minutes after the neurosecretory cells, and the timing of onset of these two activities appeared to be strictly regulated by a neural mechanism. The implication of the coordinated activity for development and maturation of imaginal tissues, including the flight motor system, and possible functions of the neuropeptides in this development are discussed.

Sply regulation of sphingolipid signaling molecules is essential for Drosophila development.

Sphingosine-1-phosphate is a sphingolipid metabolite that regulates cell proliferation, migration and apoptosis through specific signaling pathways. Sphingosine-1-phosphate lyase catalyzes the conversion of sphingosine-1-phosphate to ethanolamine phosphate and a fatty aldehyde. We report the cloning of the Drosophila sphingosine-1-phosphate lyase gene (Sply) and demonstrate its importance for adult muscle development and integrity, reproduction and larval viability. Sply expression is temporally regulated, with onset of expression during mid-embryogenesis. Sply null mutants accumulate both phosphorylated and unphosphorylated sphingoid bases and exhibit semi-lethality, increased apoptosis in developing embryos, diminished egg-laying, and gross pattern abnormalities in dorsal longitudinal flight muscles. These defects are corrected by restoring Sply expression or by introduction of a suppressor mutation that diminishes sphingolipid synthesis and accumulation of sphingolipid intermediates. This is the first demonstration of novel and complex developmental pathologies directly linked to a disruption of sphingolipid catabolism in metazoans.

Stage-specific activity patterns affect motoneuron axonal retraction and outgrowth during the metamorphosis of Manduca sexta.

During the metamorphosis of holometabolous insects, most larval muscles and sensory neurons are replaced by new adult elements, whereas most motoneurons persist and are remodelled to serve new adult functions. In Manduca sexta, the formation of the anlagen of the adult dorsal longitudinal flight muscle (DLM) is characterized by retraction of axonal terminals and dendrites of persisting larval motoneurons, partial target muscle degeneration and myoblast accumulation during late larval life. Most of these structural changes have been attributed to hormonal control, not only because ecdysteroids govern metamorphosis, but also because motoneurons express ecdysteroid receptors and experimental manipulations of ecdysteroid titres perturb normal development. To test whether activity-dependent mechanisms also came into play, chronic extracellular recordings were conducted in vivo from the five future DLM motoneurons throughout the last 3 days of larval life. Motoneuron activity is regulated developmentally. The types of motoneurons recruited, the number of motor spikes and the duration of bursts change in a stereotypical fashion during different stages, indicating an internal control of motor activity. A characteristic cessation in the activity of the five future DLM motoneurons coincides in time with the retraction of their dendrites and their terminal arborizations, whereas their activation during ecdysis coincides with the onset of new outgrowth. Inducing advanced activity by stimulating the motoneurons selectively with ecdysis-like patterns results in significant outgrowth of their terminal arborizations. Therefore, steroids might act in concert with activity-dependent mechanisms during the postembryonic modifications of neuromuscular systems.

Neural- and endocrine control of flight muscle degeneration in the adult cricket, Gryllus bimaculatus

Neural- and endocrine mechanisms controlling degeneration of a dorsal longitudinal flight muscle, M112a, have been studied in adult Gryllus bimaculatus (Orthoptera: Gryllidae). Decapitation completely prevented muscle degeneration. Implantation of a pair of corpora allata or injection of juvenile hormone III into decapitated crickets caused muscle degeneration. Denervation of M112a resulted in reduction of muscle mass compared with that in sham-operated crickets. Denervation of M112a in decapitated crickets, however, did not affect muscle mass. Birefringence and ultrastructure of M112a showed an obvious regional difference in the onset of degeneration. Fibrillar structures of M112a always disappeared from the ventral to dorsal part. Distribution of axon terminals of motor neurons and mechanical responses to the motor nerve stimuli showed that M112a is composed of five motor units with similar twitch properties. When M112a was fully denervated, regional differences in degeneration disappeared. Partial denervation resulted in denervated muscle fibers losing birefringence earlier than innervated fibers. These results suggest that juvenile hormone causes breakdown of flight muscles, and neural factors control degeneration of flight muscles to some extent under the presence of the juvenile hormone.

Postembryonic development of the dorsal longitudinal flight muscle and its innervation in Manduca sexta.

The neuromuscular systems of holometabolous insects must be remodeled during metamorphosis to allow striking behavioral changes, such as the acquisition of flight. The fast contracting dorsal longitudinal flight muscle (DLM) of Manduca arises from an anlage containing both remnants of specific larval dorsal body wall muscles and extrinsic myoblasts. In the mesothorax, the DLM is innervated by five persisting larval motoneurons: one in the mesothoracic and four in the prothoracic ganglion. These motoneurons innervate two slowly contracting body wall muscles in the larva. 2 days before pupation, the DLM template fibers begin to degenerate, whereas other muscles remain intact until pupation. Correspondingly, the motor terminals retract from the template fibers while they remain on other muscle fibers until pupation. Accumulation and proliferation of putative myoblasts also starts 2 days before pupation in close spatial relationship to the retracted motor tufts around the degenerating larval template fibers. Proliferation increases through the early pupal stages, and is detected within the anlage until the ninth day after pupation. 2 days after pupation, the anlage splits into five bundles, each innervated by one motoneuron. Striations occur on the seventh day after pupation when the growing motor axons reach the attachment sites. Subsequently, the muscle grows in volume and higher-order motor branches are formed. Within the central nervous system, there is dramatic regression of larval dendrites followed by growth of new dendrites as the persistent motoneurons assume their new role in flight behavior. Both central and peripheral remodeling follow similar time courses.

Development of the giant fiber neuron ofDrosophila melanogaster

The giant fiber system (GFS) of Drosophila melanogaster provides a convenient system in which to study neural development. It mediates escape behaviour through a small number of neurons, including the giant fibers (GFs), to innervate the tergotrochantral jump muscle (TTM) and the dorsal longitudinal flight muscles. The GFS has been intensively studied physiologically in both wild-type and mutant flies, and is often used as a system to study the effects of neural mutations on the physiology of the adult nervous system. Recently, much information has been gleaned as to how and when synaptogenesis, with its major target neurons, is achieved. However, little is known of the earlier development of this neuron. Here we have used an enhancer-trap, marking parts of the GFS, in conjunction with BrdU labelling, to attempt to follow the birth, axonogenesis, and the early morphological meeting of the GFs with their target neurons. From these anatomical observations we propose that the GF cell is not born during the larval or pupal stages and, therefore. appears to be a persistent embryonic cell. The axons of the GFs develop during the third instar. During the early pupal stages the GFs contact other identified neurons of the GFS. In addition, we see some aberrant development of the network, with some flies carrying only one GF, and yet others with extended axons. We present a model for the initial joining of the GFs and tergotrochanteral motorneurons (TTMns).

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.

Patterning the dorsal longitudinal flight muscles (DLM) of Drosophila: insights from the ablation of larval scaffolds.

The six Dorsal Longitudinal flight Muscles (DLMs) of Drosophila develop from three larval muscles that persist into metamorphosis and serve as scaffolds for the formation of the adult fibers. We have examined the effect of muscle scaffold ablation on the development of DLMs during metamorphosis. Using markers that are specific to muscle and myoblasts we show that in response to the ablation, myoblasts which would normally fuse with the larval muscle, fuse with each other instead, to generate the adult fibers in the appropriate regions of the thorax. The development of these de novo DLMs is delayed and is reflected in the delayed expression of erect wing, a transcription factor thought to control differentiation events associated with myoblast fusion. The newly arising muscles express the appropriate adult-specific Actin isoform (88F), indicating that they have the correct muscle identity. However, there are frequent errors in the number of muscle fibers generated. Ablation of the larval scaffolds for the DLMs has revealed an underlying potential of the DLM myoblasts to initiate de novo myogenesis in a manner that resembles the mode of formation of the Dorso-Ventral Muscles, DVMs, which are the other group of indirect flight muscles. Therefore, it appears that the use of larval scaffolds is a superimposition on a commonly used mechanism of myogenesis in Drosophila. Our results show that the role of the persistent larval muscles in muscle patterning involves the partitioning of DLM myoblasts, and in doing so, they regulate formation of the correct number of DLM fibers.