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.     

Mechanical analysis of Drosophila indirect flight and jump muscles

The genetic advantages of Drosophila make it a very appealing choice for investigating muscle development, muscle physiology and muscle protein structure and function. To take full advantage of this model organism, it has been vital to develop isolated Drosophila muscle preparations that can be mechanically evaluated. We describe techniques to isolate, prepare and mechanically analyze skinned muscle fibers from two Drosophila muscle types, the indirect flight muscle and the jump muscle. The function of the indirect flight muscle is similar to vertebrate cardiac muscle, to generate power in an oscillatory manner. The indirect flight muscle is ideal for evaluating the influence of protein mutations on muscle and cross-bridge stiffness, oscillatory power, and deriving cross-bridge rate constants. Jump muscle physiology and structure are more similar to skeletal vertebrate muscle than indirect flight muscle, and it is ideal for measuring maximum shortening velocity, force-velocity characteristics and steady-state power generation.     

Stereotaxic map of the muscle fibers in the indirect flight muscles of Drosophila melanogaster

It is possible to monitor the electrical activity of the motor neurons of Drosophila by recording the electrical activity of the muscle fibers. We have found that it is possible to specify the location of the subcuticular terminations and to describe the orientation within the thorax for the individual muscle fibers, because of the large size of the fibers and because the surface anatomy of Drosophila is known in detail. A map has been made to indicate the location of the muscle fibers with respect to superficial landmarks. The importance of the stereotaxic map for physiological studies is discussed.     

A nonsense mutation within the act88F actin gene disrupts myofibril formation in Drosophila indirect flight muscles

We have investigated the molecular basis of muscle abnormalities in the flightless Drosophila mutant lfm(3)7. This EMS-induced, semi-dominant allele was isolated by Mogami and Hotta (1981) and was shown to disrupt the organization of myofibrils in indirect flight muscles. Here we demonstrate that lfm(3)7 contains a nonsense mutation within codon 355 of the act88F actin gene. A single G greater than A transition converts a tryptophan (TGG) codon to an opal (TGA) terminator, thus deleting the carboxy-terminal 20 amino acids of an actin isoform that accumulates only in thoracic flight muscles. The truncated actin polypeptide is stable, and retains antigenicity to at least two anti-Drosophila actin monoclonal antibodies. We suggest that abnormalities in lfm(3)7 flight muscles result from incorporation of the mutant actin isoform into assembling myofibrils.     

Protein synthesis during flight muscle development in normal and wupB indirect flight muscles of Drosophila melanogaster

We have looked at protein synthesis in Drosophila pupae during normal and abnormal development of indirect flight muscle. Abnormal development was followed in the dominant flightless mutant wupB isolated by Hotta and Benzer (Genetic Mechanisms of Development, pp. 129–167. Academic Press, New York, 1972). The mutant muscles in adult wupB flies have abnormal morphology and disorganized myofibrils. Protein synthesis in developing muscle was followed on SDS-polyacrylamide gels. During early stages of development (55–60 h) protein synthesis patterns are similar in the mutant and the wild-type. However, at 61 h, the mutant shows a transient increase in synthesis of the 68 and 70 kDa heat shock proteins. This is followed at about 70 h by a divergence of the patterns of synthesis of other proteins seen in the mutant and wild type. These results suggest that induction of heat shock protein synthesis is an early event in abnormal morphogenesis in this mutant.     

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.     

Development of ion channels in the flight muscles of Drosophila

An entire picture of developing membrane electrical properties can be observed in the flight muscles (DLM) of Drosophila. The developmental history of membrane electrogenesis begins in the mid-pupal period and extends into the second day of adult life. Of five prominent extra-junctional ion currents which can be observed, only two are clearly mature before the adult ecloses from the pupal case. These are the two voltage-activated potassium currents, a fast transient current, and a slowly activating current. A fast transient calcium current rapidly develops around the time of adult eclosion. Suprisingly, two more ion currents develop in the adult stage: a fast transient Ca2+-activated potassium current develops during the first few hours of adult life, and a slow noninactivating inward current develops during the following two days. Both the earlier and later developing potassium currents of the transient type function in the role of fast spike repolarization in the adult. However, the later developing current appears to largely supplant the earlier developing current in this role. Thus, Shaker mutants which specifically lack the earlier developing K+ current, nevertheless, have normal appearing action potentials in mature muscle cells.     

Giant fiber activation of flight muscles in Drosophila: asynchrony in latency of wing depressor fibers

In Drosophila, brain stimulation of the giant fiber pathway brings about highly stereotyped electrical responses in target muscles involved in the escape response. Both the order of muscle response and the latency of that response are predictable in wild-type flies. The neuronal circuit to the targets is well defined and has been used in the analysis of a number of mutant phenotypes, including induced anomalies in temperature-sensitive (ts) mutations such as shibire (shi). It has been assumed that the stereotyped response includes simultaneous activation of all six fibers of the wing depressor muscle, DLM, resulting in equal latencies for all fibers. We report here a small, but distinct, inherent difference in latency between two sets of DLM fibers in a proportion of two wild-type strains as well as in a strain carrying the ts mutation shi. This difference may occur on one or both sides of an individual, is stable over time, and persists when the motor axon is stimulated peripherally. These results, due to the circuit leading to the target, suggest that the difference in latency arises peripherally. In flies reared at the shi permissive temperature (22 degrees C), the difference is more common in shi than in wild-type flies; however, in shi flies reared at 18 degrees C, the prevalence resembles that of wild-type flies. This indicates a subtle expression of the shi defect even at the presumed permissive temperature of 22 degrees C. The difference in latency is similar to that induced in shi flies whose development is affected by pupal heat pulse. Thus, correct interpretation of differences in latency, e.g., in shi/wild-type mosaic flies or in flies with mutations affecting the GF pathway, requires recognition of the inherent asynchrony that can occur between DLM fibers.     

Screen for new mutations on the 2nd chromosome involved in indirect flight muscle development in Drosophila melanogaster.

An extensive ethylmethanesulfonate mutagenesis of Drosophila melanogaster was undertaken to isolate the stronger alleles of 3 indirect flight-muscle mutations. We isolated 17 strong mutant lines, with nearly complete penetrance and expressivity, using direct screening under polarized light, from more than 1700 mutagenized chromosomes. On complementation, we found 11 of these 17 mutant lines to be alleles of 3 indirect flight-muscle mutations (Ifm(2)RU1, 3 noncomplementing lines; ifm(2)RU2, 6 alleles; ifm(2)RU3, 2 alleles) of the previously isolated 8 complementation groups (Ifm(2)RU1to ifm(2)RU8). In addition, we found 6 new complementation groups with strong defects in adult-muscle morphology; we named these ifm(2)RS1 to ifm(2)RS6. All mutant lines were mapped by meiotic recombination, and 5 of the 6 new complementation lines were mapped using chromosome deficiencies. ifm(2)RS1 maps to a region that harbors ifm(2)RU4 (a mutation that was isolated previously); however, theses are not alleles because each complements the other mutation, and the mutant-muscle phenotype is very different. We used direct screening under polarized light to find recessive mutations; although this method was labor intensive, it can be used to identify recessive genes involved in myogenesis, unlike screens for flightlessness or wing-position defects. This screen identifies regions on the second chromosome that harbor probable genes that are likely expressed in the mesoderm and are thought to be involved in myogenesis. This screen has generated valuable resources that will help us to understand the role of many molecular players involved in myogenesis.     

Aging enhances indirect flight muscle fiber performance yet decreases flight ability in Drosophila

We investigated the effects of aging on Drosophila melanogaster indirect flight muscle from the whole organism to the actomyosin cross-bridge. Median-aged (49-day-old) flies were flight impaired, had normal myofilament number and packing, barely longer sarcomeres, and slight mitochondrial deterioration compared with young (3-day-old) flies. Old (56-day-old) flies were unable to beat their wings, had deteriorated ultrastructure with severe mitochondrial damage, and their skinned fibers failed to activate with calcium. Small-amplitude sinusoidal length perturbation analysis showed median-aged indirect flight muscle fibers developed greater than twice the isometric force and power output of young fibers, yet cross-bridge kinetics were similar. Large increases in elastic and viscous moduli amplitude under active, passive, and rigor conditions suggest that median-aged fibers become stiffer longitudinally. Small-angle x-ray diffraction indicates that myosin heads move increasingly toward the thin filament with age, accounting for the increased transverse stiffness via cross-bridge formation. We propose that the observed protein composition changes in the connecting filaments, which anchor the thick filaments to the Z-disk, produce compensatory increases in longitudinal stiffness, isometric tension, power and actomyosin interaction in aging indirect flight muscle. We also speculate that a lack of MgATP due to damaged mitochondria accounts for the decreased flight performance.