Larval Body Wall Research Articles

Actions of MDMA at glutamatergic neuromuscular junctions.

3,4-Methylenedioxymethamphetamine (MDMA, "Ecstasy") compels mammalian serotonergic neurons to release serotonin (5-HT). In this study, MDMA altered synaptic transmission presynaptically by enhancing quantal release in two model glutamatergic synapses-the neuromuscular junction (NMJ) of the crayfish opener muscle, which is enhanced by exogenous 5-HT application, and the NMJ of a larval body wall muscle in Drosophila melanogaster, which is insensitive to exogenous 5-HT application. At the crayfish NMJ, MDMA mimicked the actions of 5-HT but only at a substantially higher concentration. At the Drosophila NMJ, MDMA altered synaptic transmission but not through a 5-HT receptor. Using simple invertebrate preparations, we have demonstrated an additional non-serotonergic mechanism of MDMA activity that has not yet been addressed in vertebrate systems and that may play an important role in understanding the mechanism of action for a commonly abused drug.

Enhanced Locomotion Caused by Loss of the Drosophila DEG/ENaC Protein Pickpocket1

Coordination of rhythmic locomotion depends upon a precisely balanced interplay between central and peripheral control mechanisms [1]. Although poorly understood, peripheral proprioceptive mechanosensory input is thought to provide information about body position for moment-to-moment modifications of central mechanisms mediating rhythmic motor output [2]. Pickpocket1 (PPK1) is a Drosophila subunit of the epithelial sodium channel (ENaC) family displaying limited expression in multiple dendritic (md) sensory neurons tiling the larval body wall and a small number of bipolar neurons in the upper brain [3]. ppk1 null mutant larvae had normal external touch sensation and md neuron morphology but displayed striking alterations in crawling behavior. Loss of PPK1 function caused an increase in crawling speed and an unusual straight path with decreased stops and turns relative to wild-type. This enhanced locomotion resulted from sustained peristaltic contraction wave cycling at higher frequency with a significant decrease in pause period between contraction cycles. The mutant phenotype was rescued by a wild-type PPK1 transgene and duplicated by expressing a ppk1RNAi transgene or a dominant-negative PPK1 isoform. These results demonstrate that the PPK1 channel plays an essential role in controlling rhythmic locomotion and provide a powerful genetic model system for further analysis of central and peripheral control mechanisms and their role in movement disorders.

Characterization of development, behavior and neuromuscular physiology in the phorid fly, Megaselia scalaris

The Phoridae is known as ‘scuttle flies’ because they walk in rapid bursts of movement with short pauses between. In this study, larval locomotive behavior and development was characterized in the phorid, Megaselia scalaris. Comparison was made with the well-characterized fruit fly model, Drosophila melanogaster. Developmentally, the rate of maturation was consistently slower for Megaselia than Drosophila. This disparity was exaggerated at lower temperatures, particularly during larval development. In addition to slower growth, movements in Megaselia were also slower, as evidenced by reduced rates of larval body wall contractions and mouth hook movements. Megaselia larvae also displayed a unique behavior of swallowing air when exposed to a small pool of liquid. This permitted floating upon immersion and, therefore, might prevent drowning in the natural environment. The anatomical and physiological properties of a neuromuscular junction in the phorid larvae were also examined. The innervation of the motor nerve terminals on the ventral abdominal muscle (m6) is innervated by Type Ib and Is axons, similar to Drosophila. As in Drosophila, the Is terminals produce larger excitatory postsynaptic potentials (EPSPs) than the Ib. The amplitudes of the EPSPs in M. scalaris were reduced compared to those of D. melanogaster, but unlike D. melanogaster the EPSPs showed marked facilitation when stimulated with a 20 Hz train. We conclude that there may be differences in synaptic structure of the nerve terminals that could account for the different electrophysiological behaviors.

Generation of GAL4-responsive muscleblind constructs.

The muscleblind (mbl) gene encodes protein isoforms Mbl A to Mbl D, which arise by alternative splicing from a common primary transcript. Mbl A, B, and C contain two Zn-finger domains of the type Cys3His, while Mbl D contains only one complete Zn finger. Loss of function mutations in the gene reveal that mbl is involved in both terminal photoreceptor and muscle differentiation in Drosophila. During retina development mbl is essential for rhabdomere differentiation in photoreceptor neuron. Clones homozygous null for mbl completely lack these lightharvesting structures (Begemann et al., 1997). Similarly, the terminal differentiation of the larval body wall muscles is compromised in mbl mutant embryos. In these mutant embryos muscles show sarcomeres with disorganized Z-lines and reduction in the extracellular matrix of indirect muscle attachments (Artero et al., 1998). In addition, biochemical studies aimed at understanding the molecular basis of myotonic dystrophy (DM), a dominant-autosomic disease, identified the human mbl paralogs MBNL and MBLL as critical elements in the DM pathogenesis pathway (Miller et al., 2000). Despite intense research, however, the molecular function of Mbl proteins has not yet been elucidated. The current working models point to a role for MBNL/MBLL proteins in RNA metabolism, possibly during pre-mRNA splicing or mRNA nuclear export. These models are supported mainly by the observation that MBNL/MBLL proteins bind to CUG and CCUG expansions (Miller et al., 2000; Mankodi et al., 2001; Fardaei et al., 2002). To generate both gain of function defects in the fly as well as genetic tools to use in a Drosophila model for the human disease, we made three UAS constructs containing the Drosophila protein isoforms Mbl A and Mbl C and the human MBNL protein isoform encoded by the cDNA KIAA0428, which includes most of the alternatively used exons in the MBNL locus (Fardaei et al., 2002). UAS constructs (Fig. 1) were injected into fly embryos of the genotype white using standard germline transformation methods (Spradling and Rubin, 1982). We established five independent transgenic lines carrying UAS-mbl A, six transgenic lines carrying UAS-mbl C, and three transgenic lines carrying UAS-KIAA0428. These lines were mapped by genetic crosses to the indicated chromosomes (Fig. 1). Direct experimental evidence of Gal4-driven expression for these constructs was obtained by crossing UAS-mbl A and UAS-mbl C to the engrailed-Gal4 driver, which gives a segment polarity pattern of expression clearly different from the endogenous mbl transcription. In situ hybridization in these embryos clearly detected ectopic mbl expression driven by the engrailed promoter (Fig. 2A–C). In addition, expression of the transgenes was targeted to the precursors of adult structures to reveal morphological defects indicative of their biological activity. Targeted expression of Mbl A and Mbl C to the Drosophila eye imaginal disc driven by an endogenous sevenless promoter (sev-Gal4) led to a rough eye phenotype which ranged from a very mild defect in the case of UAS-mbl A to a clearly rough eye in the case of UAS-mbl C (Fig. 2D–F). Expression of UAS-KIAA0428 under similar conditions led to a severely rough eye phenotype and lethality, depending on the transgene used. We used our weakest transgene (UAS-KIAA0428.KD.1) and an incubation temperature of 25°C for these crosses in order to obtain viable offspring and a milder eye phenotype (Fig. 2G). Tangential sections through these rough eyes revealed morphological defects at the cellular level. In all cases we detected ommatidia showing typical planar cell polarity (PCP) defects but also, especially when the UAS-mbl C was overexpressed, photoreceptor loss. In a similar set of experiments, we targeted expression of all three transgenes to the wing imaginal disc using 69B-Gal4 or apterous-Gal4 as drivers (Fig. 2H–K). Offspring from these crosses revealed defects in the mor-

Tiling of the Drosophila epidermis by multidendritic sensory neurons.

Insect dendritic arborization (da) neurons provide an opportunity to examine how diverse dendrite morphologies and dendritic territories are established during development. We have examined the morphologies of Drosophila da neurons by using the MARCM (mosaic analysis with a repressible cell marker) system. We show that each of the 15 neurons per abdominal hemisegment spread dendrites to characteristic regions of the epidermis. We place these neurons into four distinct morphological classes distinguished primarily by their dendrite branching complexities. Some class assignments correlate with known proneural gene requirements as well as with central axonal projections. Our data indicate that cells within two morphological classes partition the body wall into distinct, non-overlapping territorial domains and thus are organized as separate tiled sensory systems. The dendritic domains of cells in different classes, by contrast, can overlap extensively. We have examined the cell-autonomous roles of starry night (stan) (also known as flamingo (fmi)) and sequoia (seq) in tiling. Neurons with these genes mutated generally terminate their dendritic fields at normal locations at the lateral margin and segment border, where they meet or approach the like dendrites of adjacent neurons. However, stan mutant neurons occasionally send sparsely branched processes beyond these territories that could potentially mix with adjacent like dendrites. Together, our data suggest that widespread tiling of the larval body wall involves interactions between growing dendritic processes and as yet unidentified signals that allow avoidance by like dendrites.

A conditional tissue-specific transgene expression system using inducible GAL4.

In Drosophila, the most widely used system for generating spatially restricted transgene expression is based on the yeast GAL4 protein and its target upstream activating sequence (UAS). To permit temporal as well as spatial control over UAS-transgene expression, we have explored the use of a conditional RU486-dependent GAL4 protein (GeneSwitch) in Drosophila. By using cloned promoter fragments of the embryonic lethal abnormal vision gene or the myosin heavy chain gene, we have expressed GeneSwitch specifically in neurons or muscles and show that its transcriptional activity within the target tissues depends on the presence of the activator RU486 (mifepristone). We used available UAS-reporter lines to demonstrate RU486-dependent tissue-specific transgene expression in larvae. Reporter protein expression could be detected 5 h after systemic application of RU486 by either feeding or "larval bathing." Transgene expression levels were dose-dependent on RU486 concentration in larval food, with low background expression in the absence of RU486. By using genetically altered ion channels as reporters, we were able to change the physiological properties of larval bodywall muscles in an RU486-dependent fashion. We demonstrate here the applicability of GeneSwitch for conditional tissue-specific expression in Drosophila, and we provide tools to control pre- and postsynaptic expression of transgenes at the larval neuromuscular junction during postembryonic life.

EsMlp, a Muscle-LIM Protein Gene, Is Up-regulated during Cold Exposure in the Freeze-Avoiding Larvae of Epiblema scudderiana

Screening of a cDNA library identified transcripts that were up-regulated by cold (4 or −20°C) exposure in larvae of the freeze-avoiding goldenrod gall moth, Epiblema scudderiana. One clone contained a full-length open reading frame encoding a protein of 94 amino acids. The gene product, with 79.1% of residues identical with the Drosophila LIM protein Mlp60A, was named EsMlp and contained a single LIM domain and consensus sequences characteristic of a LIM protein. Transcript levels rose approx twofold when larvae were shifted from 4 to −20°C and approx threefold over the midwinter months compared with larvae sampled in October or April. EsMlp expression was high in larval head (possibly due to expression in pharyngeal muscles) and body wall but was not detected in fat body. Immunoblotting revealed a three- to fourfold increase in EsMlp protein in midwinter larvae (January-February) compared with November-collected animals and a further rise to eightfold higher than November values in larvae collected in April. Cold up-regulation of EsMlp and the pattern of EsMlp levels in the larvae suggest possible roles for the protein, such as in muscle maintenance over the winter or as a preparative function that could facilitate the rapid resumption of development and metamorphosis when environmental temperatures rise in the spring.

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.

The ryanodine receptor is essential for larval development in Drosophila melanogaster.

We have investigated the role of the ryanodine receptor in Drosophila development by using pharmacological and genetic approaches. We identified a P element insertion in the Drosophila ryanodine receptor gene, Ryanodine receptor 44F (Ryr), and used it to generate the hypomorphic allele Ryr(16). An examination of hypodermal, visceral, and circulatory muscle showed that, in each case, muscle contraction was impaired in Ryr(16) larvae. Treatment with the drug ryanodine, a highly specific modulator of ryanodine receptor channel activity, also inhibited muscle function, and, at high levels, completely blocked hypodermal muscle contraction. These results suggest that the ryanodine receptor is required for proper muscle function and may be essential for excitation-contraction coupling in larval body wall muscles. Nonmuscle roles of Ryr were also investigated. Ryanodine-sensitive Ca(2+) stores had previously been implicated in phototransduction; to address this, we generated Ryr(16) mutant clones in the adult eye and performed whole-cell, patch-clamp recordings on dissociated ommatidia. Our results do not support a role for Ryr in normal light responses.

Peripheral distribution of presynaptic sites of abdominal motor and modulatory neurons in Manduca sexta larvae.

Insect muscle fibers are commonly innervated by multiple motor neurons and efferent unpaired median (UM) neurons. The role of UM neurons in the modulation rather than rapid activation of muscle contraction (Evans and O'Shea [1977] Nature 270:257-259) suggests that their terminal varicosities may differ structurally and functionally from the presynaptic terminals of motor neurons. Furthermore, differences in the characteristics of UM neuron terminal varicosities may be correlated with functional differences among their diverse target muscles. Larval abdominal body wall muscles in the hawkmoth, Manduca sexta, consist of large, elongated fibers that are multiterminally innervated by one and occasionally two motor neurons (Levine and Truman [1985] J. Neurosci. 5:2424-2431). The fibers are also innervated by one of two efferent UM neurons that bifurcate to innervate targets on both sides of the abdomen (Pflüger et al. [1993] J. Comp. Neurol. 335:508-522). In this study, the intracellular tracer biocytin was used to identify the targets of the UM neurons and to distinguish their terminal axonal varicosities on the muscle fibers. An antiserum to the synaptic vesicle protein, synaptotagmin, was used to label synaptic vesicles, and the styryl dye FM1-43 was used to demonstrate release and recycling. Most of the abdominal muscles in a given hemisegment were found to be supplied by one of the two UM neurons. Terminal varicosities of the excitatory motor neurons were large (3-7 pm) and were found in rows of rosettes that extended to every aspect of the muscle fiber; these varicosities were designated as type I terminals. The UM neuron terminal varicosities also occupied every aspect of the fiber but were smaller (1-3 microm) and more separated from each other; these were designated as type II terminals. Both type I and type II terminals are synaptotagmin immunoreactive and, as shown by FM1-43 staining, are sites of synaptic vesicle recycling. The excitatory motor neuron terminals (type I) could easily be loaded and unloaded with FM1-43, which indicates their capacity for repeated vesicular exocytosis and recycling. In contrast, the dye could not as readily be unloaded from UM neuron terminals (type II), which may indicate that they have a slower turnover of synaptic vesicles.

Mutational Analysis of the Shab-encoded Delayed Rectifier K+ Channels in Drosophila

K(+) currents in Drosophila muscles have been resolved into two voltage-activated currents (I(A) and I(K)) and two Ca(2+)-activated currents (I(CF) and I(CS)). Mutations that affect I(A) (Shaker) and I(CF) (slowpoke) have helped greatly in the analysis of these currents and their role in membrane excitability. Lack of mutations that specifically affect channels for the delayed rectifier current (I(K)) has made their genetic and functional identity difficult to elucidate. With the help of mutations in the Shab K(+) channel gene, we show that this gene encodes the delayed rectifier K(+) channels in Drosophila. Three mutant alleles with a temperature-sensitive paralytic phenotype were analyzed. Analysis of the ionic currents from mutant larval body wall muscles showed a specific effect on delayed rectifier K(+) current (I(K)). Two of the mutant alleles contain missense mutations, one in the amino-terminal region of the channel protein and the other in the pore region of the channel. The third allele contains two deletions in the amino-terminal region and is a null allele. These observations identity the channels that carry the delayed rectifier current and provide an in vivo physiological role for the Shab-encoded K(+) channels in Drosophila. The availability of mutations that affect I(K) opens up possibilities for studying I(K) and its role in larval muscle excitability.

Biogenic amine systems in the fruit fly Drosophila melanogaster.

Biogenic amines are important neuroactive molecules of the central nervous system (CNS) of several insect species. Serotonin (5HT), dopamine (DA), histamine (HA), and octopamine (OA) are the amines which have been extensively studied in Drosophila melanogaster. Each one of the four aminergic neuronal systems exhibits a stereotypic pattern of a small number of neurons that are widely distributed in the fly CNS. In this review, histochemical and immunocytochemical data on the distribution of the amine neurons in the larval and adult nervous system, are summarized. The majority of DA and 5HT neurons are interneurons, most of which are found in bilateral clusters. 5HT innervation is found in the feeding apparatus as well as in the endocrine organ of the larva, the ring gland. The octopaminergic neuronal population consists of both interneurons and efferent neurons. In the larval CNS all OA immunoreactive somata are localized in the midline of the ventral ganglion while in the adult CNS both unpaired neurons and bilateral clusters of immunoreactive cells are observed. One target of OA innervation is the abdominal muscles of the larval body wall where OA immunoreactivity is associated with the type II boutons in the axonal terminals. Histamine is mainly found in all photoreceptor cells where it is considered to be the major neurotransmitter molecule, and in specific mechanosensory neurons of the peripheral nervous system. Similarities between specific aminergic neurons and innervation sites in Drosophila and in other insect species are discussed. In addition, studies on the development and differentiation of 5HT and DA neurons are reviewed and data on the localization of 5HT, DA, and OA receptors are included as well. Finally, an overview on the isolation of the genes and the mutations in the amine biosynthetic pathways is presented and the implications of the molecular genetic approach in Drosophila are discussed.

Respecified larval proleg and body wall muscles circulate hemolymph in developing wings of Manduca sexta pupae.

Most larval external muscles in Manduca sexta degenerate at pupation, with the exception of the accessory planta retractor muscles (APRMs) in proleg-bearing abdominal segment 3 and their homologs in non-proleg-bearing abdominal segment 2. In pupae, these APRMs exhibit a rhythmic 'pupal motor pattern' in which all four muscles contract synchronously at approximately 4 s intervals for long bouts, without externally visible movements. On the basis of indirect evidence, it was proposed previously that APRM contractions during the pupal motor pattern circulate hemolymph in the developing wings and legs. This hypothesis was tested in the present study by making simultaneous electromyographic recordings of APRM activity and contact thermographic recordings of hemolymph flow in pupal wings. APRM contractions and hemolymph flow were strictly correlated during the pupal motor pattern. The proposed circulatory mechanism was further supported by the findings that unilateral ablation of APRMs or mechanical uncoupling of the wings from the abdomen essentially abolished wing hemolymph flow on the manipulated side of the body. Rhythmic contractions of intersegmental muscles, which sometimes accompany the pupal motor pattern, had a negligible effect on hemolymph flow. The conversion of larval proleg and body wall muscles to a circulatory function in pupae represents a particularly dramatic example of functional respecification during metamorphosis.

Ionic Currents in Larval Muscles of Drosophila

Publisher Summary The larval muscle preparation provides an excellent in situ assay system for the studies of ion channels in Drosophila . Larval body wall muscles of Drosophila provide a very useful system for studying K + and Ca 2+ currents. This chapter demonstrates that the function of the native K + and Ca 2+ channels can be analyzed with the advanced biophysical and pharmacological techniques that have been applied to other systems. The physiological and biophysical properties of ion channels can be analyzed from the single channel to the whole cell level, and the functional roles of different classes of ion channels can be directly elucidated in Drosophila muscles because of the available mutants. The chapter discusses the subunit composition of the channels, the genes encoding the subunits, the kinetic properties, and voltage dependency of the currents, pharmacological blockers and activators, and the signal transduction pathways regulating or modulating the channel properties. The physiological aspects of the currents are presented. Muscle currents in adults are briefly summarized for comparison with currents in the larval muscles. The role of different ionic currents in the physiology of muscle fibers is discussed in experiments involving current clamping and membrane potential measurements.

Development of Larval Body Wall Muscles

Publisher Summary Larval muscles in Drosophila form a complex pattern of contractile fibers that attach to the inner surface of the developing epidermis. Each segment has its own, highly specific set of muscles, but from the point of view of the neuromuscular junction, most attention has been focused on the muscles of the abdominal segments. The formation of the muscle pattern depends on an interaction between a general pathway of myogenic differentiation and local controls that endow individual muscles with their special characteristics. Each muscle synthesizes a contractile apparatus and anchorages to the epidermis and forms a neuromuscular junction with the terminals of innervating motor neurons. These functions are common to all muscles and, at least in part, are downstream of control factors such as Dmef-2 . However, each muscle in the pattern has its own highly distinctive properties, including size, shape, orientation, sites of attachment, and innervation by particular motor neurons. These individual properties depend on the local expression of transcription factors such as S59 and Kr . The combination of general and specific regulatory mechanisms in the myogenic pathway leads to the formation of a specialized set of contractile elements on which motor neurons can act to produce the coordinated movements of larval behavior.

Regulated spacing of synapses and presynaptic active zones at larval neuromuscular junctions in different genotypes of the flies Drosophila and Sarcophaga.

Synapses at larval neuromuscular junctions of the flies Drosophila melanogaster and Sarcophaga bullata are not distributed randomly. They have been studied in serial electron micrographs of two identified axons (axons 1 and 2) that innervate ventral longitudinal muscles 6 and 7 of the larval body wall. The following fly larvae were examined: axon 1--wild-type Sarcophaga and Drosophila and Drosophila mutants dunce(m14) and fasII(e76), a hypomorphic allele of the fasciclin II gene; and axon 2--drosophila wild-type, dunce(m14), and fasII(e76). These lines were selected to provide a wide range of nerve terminal phenotypes in which to study the distribution and spacing of synapses. Each terminal varicosity is applied closely to the underlying subsynaptic reticulum of the muscle fiber and has 15-40 synapses. Each synapse usually bears one or more active zones, characterized by dense bodies that are T-shaped in cross section; they are located at the presumed sites of transmitter release. The distribution of synapses was characterized from the center-to-center distance of each synapse to its nearest neighbor. The mean spacing between nearest-neighbor pairs ranged from 0.84 microm to 1.05 microm for axon 1, showing no significant difference regardless of genotype. The corresponding values for axon 2, 0.58 microm to 0.75 microm, were also statistically indistinguishable from one another in terminals of different genotype but differed significantly from the values for axon 1. Thus, the functional class of the axon provides a clear prediction of the spacing of its synapses, suggesting that spacing may be determined by the functional properties of transmission at the two types of terminals. Individual dense bodies were situated mostly at least 0.4 microm away from one another, suggesting that an interaction between neighboring active zones could prevent their final positions from being located more closely.

A Novel Lectin from Sarcophaga: ITS PURIFICATION, CHARACTERIZATION, AND cDNA CLONING

A novel C-type lectin that agglutinates rabbit red cells was purified from NIH-Sape-4 cells derived from the flesh fly (Sarcophaga peregrina), and its cDNA was isolated. This lectin, named granulocytin, appeared to be a trimer of a 20-kDa subunit consisting of 151 amino acid residues. The gene for granulocytin was activated in third instar larvae, and its expression was enhanced when the larval body wall was injured. In third instar larvae, granulocytin was found to be synthesized by hemocytes and secreted into the hemolymph. The molecular mass and gene expression patterns of granulocytin were very similar to those of Drosophila lectin that we reported previously (Haq, S., Kubo, T., Kurata, S., Kobayashi, A., and Natori, S. (1996) J. Biol. Chem. 271, 20213-20218). However, these two lectins showed amino acid identities of 20% at most, and no significant hapten sugar for granulocytin was identified.

18 Patch-Clamp Recordings from Drosophila Presynaptic Terminals

Publisher Summary This chapter focuses on patch-clamp recordings from drosophila presynaptic terminals. In general, the events that take place in synaptic terminals have had to be inferred from recordings performed in the soma. However, it is generally admitted that the synaptic membrane is endowed with a wide and specialized repertoire of ion channels whose activity determines the modulation of neural signaling. Also, mounting evidence from the in situ expression of synaptic proteins and, most relevant, localization of specific protein isoforms indicates that synapses are very diverse. The necessity of approaching synaptic studies from a multidisciplinary perspective justifies the effort to develop suitable methods in organisms where genetic and behavioral studies are also feasible. To that end, the chapter has tried to develop a new preparation for synaptic transmission in Drosophila. The Drosophila larval body-wall muscle fibers are innervated by several axons that produce morphologically distinct synaptic contacts on specific fibers. The chapter describes several types of synaptic boutons, classed as types I to III, which differ in their size, morphology, and vesicle phenotype.

Myocyte-specific enhancer factor 2 acts cooperatively with a muscle activator region to regulate Drosophila tropomyosin gene muscle expression.

MEF2 (myocyte-specific enhancer factor 2) is a MADS box transcription factor that is thought to be a key regulator of myogenesis in vertebrates. Mutations in the Drosophila homologue of the mef2 gene indicate that it plays a key role in regulating myogenesis in Drosophila. We show here that the Drosophila tropomyosin I (TmI) gene is a target gene for mef2 regulation. The TmI gene contains a proximal and a distal muscle enhancer within the first intron of the gene. We show that both enhancers contain a MEF2 binding site and that a mutation in the MEF2 binding site of either enhancer significantly reduces reporter gene expression in embryonic, larval, and adult somatic body wall muscles of transgenic flies. We also show that a high level of proximal enhancer-directed reporter gene expression in somatic muscles requires the cooperative activity of MEF2 and a cis-acting muscle activator region located within the enhancer. Thus, mef2 null mutant embryos show a significant reduction but not an elimination of TmI expression in the body wall myoblasts and muscle fibers that are present. Surprisingly, there is little effect in these mutants on TmI expression in developing visceral muscles and dorsal vessel (heart), despite the fact that MEF2 is expressed in these muscles in wild-type embryos, indicating that TmI expression is regulated differently in these muscles. Taken together, our results show that mef2 is a positive regulator of tropomyosin gene transcription that is necessary but not sufficient for high level expression in somatic muscle of the embryo, larva, and adult.

Wingless is required for the formation of a subset of muscle founder cells during Drosophila embryogenesis

The final pattern of the Drosophila larval body wall muscles depends critically on the prior segregation of muscle founder cells. We would like to understand the underlying molecular mechanisms which ensure the precise allocation and placement of these muscle founder cells. We have begun our analysis by examining the role of the segment polarity genes, known to be involved in the patterning of the ectoderm. Mutations in only one member of this class, wingless (wg), lead to the complete loss of a subset of muscle founder cells characterised by the expression of S59. Using the GAL4-targetted expression system, we find that Wingless, a secreted glycoprotein and well characterized signalling molecule, acts directly on the mesoderm to ensure the formation of S59-expressing founder cells. Moreover, we present evidence that Wg can signal across germ layers and that, in the wild-type embryo, Wg from the ectoderm could constitute an inductive signal for the initiation of the development of a subset of somatic muscles.