wishful thinking encodes a BMP type II receptor that regulates synaptic growth in Drosophila

We conducted a large-scale screen for Drosophila mutants that have structural abnormalities of the larval neuromuscular junction (NMJ). We recovered mutations in wishful thinking (wit), a gene that positively regulates synaptic growth. wit encodes a BMP type II receptor. In wit mutant larvae, the size of the NMJs is greatly reduced relative to the size of the muscles. wit NMJs have reduced evoked excitatory junctional potentials, decreased levels of the synaptic cell adhesion molecule Fasciclin II, and synaptic membrane detachment at active zones. Wit is expressed by a subset of neurons, including motoneurons. The NMJ phenotype is specifically rescued by transgenic expression of Wit only in motoneurons. Thus, Wit appears to function as a presynaptic receptor that regulates synaptic size at the Drosophila NMJ.     

Optogenetics in the teaching laboratory: using channelrhodopsin-2 to study the neural basis of behavior and synaptic physiology in Drosophila

Here we incorporate recent advances in Drosophila neurogenetics and "optogenetics" into neuroscience laboratory exercises. We used the light-activated ion channel channelrhodopsin-2 (ChR2) and tissue-specific genetic expression techniques to study the neural basis of behavior in Drosophila larvae. We designed and implemented exercises using inexpensive, easy-to-use systems for delivering blue light pulses with fine temporal control. Students first examined the behavioral effects of activating glutamatergic neurons in Drosophila larvae and then recorded excitatory junctional potentials (EJPs) mediated by ChR2 activation at the larval neuromuscular junction (NMJ). Comparison of electrically and light-evoked EJPs demonstrates that the amplitudes and time courses of light-evoked EJPs are not significantly different from those generated by electrical nerve stimulation. These exercises introduce students to new genetic technology for remotely manipulating neural activity, and they simplify the process of recording EJPs at the Drosophila larval NMJ. Relatively little research work has been done using ChR2 in Drosophila, so students have opportunities to test novel hypotheses and make tangible contributions to the scientific record. Qualitative and quantitative assessment of student experiences suggest that these exercises help convey principles of synaptic transmission while also promoting integrative and inquiry-based studies of genetics, cellular physiology, and animal behavior.     

Interaction of cytoskeleton genes with NSF2-induced neuromuscular junction overgrowth

N-Ethylmaleimide sensitive factor (NSF) is an ATPase whose activity is important for intracellular trafficking. Previous genetic analysis of Drosophila NSF2 revealed a potential link between NSF and the actin cytoskeleton. The present study was therefore undertaken to specifically examine genetic interactions between the cytoskeleton and NSF. First, we tested for loss-of-function interaction and, indeed, we found that the combination of flies heterozygous for Act5C and NSF2 alleles led to reduced viability. Second, we expanded our gain-of-function approach to include cytoskeletal genes that were not included in our previous screen. Thirteen of 30 genes tested were found to suppress neuromuscular junction (NMJ) overgrowth. Altogether, these data support the idea that diverse NSF2 developmental and physiological phenotypes are related to disruption of the cytoskeleton and the large number of genes which can partially restore NMJ overgrowth and suggests that NSF may function near the top of the actin regulatory pathway.     

Synaptic vesicle mobility and presynaptic F-actin are disrupted in a N-ethylmaleimide-sensitive factor allele of Drosophila

N-ethylmaleimide sensitive factor (NSF) can dissociate the soluble NSF attachment receptor (SNARE) complex, but NSF also participates in other intracellular trafficking functions by virtue of SNARE-independent activity. Drosophila that express a neural transgene encoding a dominant-negative form of NSF2 show an 80% reduction in the size of releasable synaptic vesicle pool, but no change in the number of vesicles in nerve terminal boutons. Here we tested the hypothesis that vesicles in the NSF2 mutant terminal are less mobile. Using a combination of genetics, pharmacology, and imaging we find a substantial reduction in vesicle mobility within the nerve terminal boutons of Drosophila NSF2 mutant larvae. Subsequent analysis revealed a decrease of filamentous actin in both NSF2 dominant-negative and loss-of-function mutants. Lastly, actin-filament disrupting drugs also decrease vesicle movement. We conclude that a factor contributing to the NSF mutant phenotype is a reduction in vesicle mobility, which is associated with decreased presynaptic F-actin. Our data are consistent with a model in which actin filaments promote vesicle mobility and suggest that NSF participates in establishing or maintaining this population of actin.     

Drosophila S6 Kinase Like Inhibits Neuromuscular Junction Growth by Downregulating the BMP Receptor Thickveins

Synaptic connections must be precisely controlled to ensure proper neural circuit formation. In Drosophila melanogaster, bone morphogenetic protein (BMP) promotes growth of the neuromuscular junction (NMJ) by binding and activating the BMP ligand receptors wishful thinking (Wit) and thickveins (Tkv) expressed in motor neurons. We report here that an evolutionally conserved, previously uncharacterized member of the S6 kinase (S6K) family S6K like (S6KL) acts as a negative regulator of BMP signaling. S6KL null mutants were viable and fertile but exhibited more satellite boutons, fewer and larger synaptic vesicles, larger spontaneous miniature excitatory junctional potential (mEJP) amplitudes, and reduced synaptic endocytosis at the NMJ terminals. Reducing the gene dose by half of tkv in S6KL mutant background reversed the NMJ overgrowth phenotype. The NMJ phenotypes of S6KL mutants were accompanied by an elevated level of Tkv protein and phosphorylated Mad, an effector of the BMP signaling pathway, in the nervous system. In addition, Tkv physically interacted with S6KL in cultured S2 cells. Furthermore, knockdown of S6KL enhanced Tkv expression, while S6KL overexpression downregulated Tkv in cultured S2 cells. This latter effect was blocked by the proteasome inhibitor MG132. Our results together demonstrate for the first time that S6KL regulates synaptic development and function by facilitating proteasomal degradation of the BMP receptor Tkv.     

Autophagy promotes synapse development in Drosophila

Autophagy, a lysosome-dependent degradation mechanism, mediates many biological processes, including cellular stress responses and neuroprotection. In this study, we demonstrate that autophagy positively regulates development of the Drosophila melanogaster larval neuromuscular junction (NMJ). Autophagy induces an NMJ overgrowth phenotype closely resembling that of highwire (hiw), an E3 ubiquitin ligase mutant. Moreover, like hiw, autophagy-induced NMJ overgrowth is suppressed by wallenda (wnd) and by a dominant-negative c-Jun NH₂-terminal kinase (bsk^DN). We show that autophagy promotes NMJ growth by reducing Hiw levels. Thus, autophagy and the ubiquitin–proteasome system converge in regulating synaptic development. Because autophagy is triggered in response to many environmental cues, our findings suggest that it is perfectly positioned to link environmental conditions with synaptic growth and plasticity.     

Anterograde Activin Signaling Regulates Postsynaptic Membrane Potential and GluRIIA/B Abundance at the Drosophila Neuromuscular Junction

Members of the TGF-β superfamily play numerous roles in nervous system development and function. In Drosophila, retrograde BMP signaling at the neuromuscular junction (NMJ) is required presynaptically for proper synapse growth and neurotransmitter release. In this study, we analyzed whether the Activin branch of the TGF-β superfamily also contributes to NMJ development and function. We find that elimination of the Activin/TGF-β type I receptor babo, or its downstream signal transducer smox, does not affect presynaptic NMJ growth or evoked excitatory junctional potentials (EJPs), but instead results in a number of postsynaptic defects including depolarized membrane potential, small size and frequency of miniature excitatory junction potentials (mEJPs), and decreased synaptic densities of the glutamate receptors GluRIIA and B. The majority of the defective smox synaptic phenotypes were rescued by muscle-specific expression of a smox transgene. Furthermore, a mutation in actβ, an Activin-like ligand that is strongly expressed in motor neurons, phenocopies babo and smox loss-of-function alleles. Our results demonstrate that anterograde Activin/TGF-β signaling at the Drosophila NMJ is crucial for achieving normal abundance and localization of several important postsynaptic signaling molecules and for regulating postsynaptic membrane physiology. Together with the well-established presynaptic role of the retrograde BMP signaling, our findings indicate that the two branches of the TGF-β superfamily are differentially deployed on each side of the Drosophila NMJ synapse to regulate distinct aspects of its development and function.     

Independent roles of Drosophila Moesin in imaginal disc morphogenesis and hedgehog signalling

The three ERM proteins (Ezrin, Radixin and Moesin) form a conserved family required in many developmental processes involving regulation of the cytoskeleton. In general, the molecular function of ERM proteins is to link specific membrane proteins to the actin cytoskeleton. In Drosophila, loss of moesin (moe) activity causes incorrect localisation of maternal determinants during oogenesis, failures in rhabdomere differentiation in the eye and alterations of epithelial integrity in the wing imaginal disc. Some aspects of Drosophila Moe are related to the activity of the small GTPase RhoA, because the reduction of RhoA activity corrects many phenotypes of moe mutant embryos and imaginal discs. We have analysed the phenotype of moesin loss-of-function alleles in the wing disc and adult wing, and studied the effects of reduced Moesin activity on signalling mediated by the Notch, Decapentaplegic, Wingless and Hedgehog pathways. We found that reductions in Moesin levels in the wing disc cause the formation of wing-tissue vesicles and large thickenings of the vein L3, corresponding to breakdowns of epithelial continuity in the wing base and modifications of Hedgehog signalling in the wing blade, respectively. We did not observe any effect on signalling pathways other than Hedgehog, indicating that the moe defects in epithelial integrity have not generalised effects on cell signalling. The effects of moe mutants on Hedgehog signalling depend on the correct gene-dose of rhoA, suggesting that the requirements for Moesin in disc morphogenesis and Hh signalling in the wing disc are mediated by its regulation of RhoA activity. The mechanism linking Moesin activity with RhoA function and Hedgehog signalling remains to be elucidated.     

A neuropeptide signaling pathway regulates synaptic growth in Drosophila

Neuropeptide signaling is integral to many aspects of neural communication, particularly modulation of membrane excitability and synaptic transmission. However, neuropeptides have not been clearly implicated in synaptic growth and development. Here, we demonstrate that cholecystokinin-like receptor (CCKLR) and drosulfakinin (DSK), its predicted ligand, are strong positive growth regulators of the Drosophila melanogaster larval neuromuscular junction (NMJ). Mutations of CCKLR or dsk produced severe NMJ undergrowth, whereas overexpression of CCKLR caused overgrowth. Presynaptic expression of CCKLR was necessary and sufficient for regulating NMJ growth. CCKLR and dsk mutants also reduced synaptic function in parallel with decreased NMJ size. Analysis of double mutants revealed that DSK/CCKLR regulation of NMJ growth occurs through the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cAMP response element binding protein (CREB) pathway. Our results demonstrate a novel role for neuropeptide signaling in synaptic development. Moreover, because the cAMP-PKA-CREB pathway is required for structural synaptic plasticity in learning and memory, DSK/CCKLR signaling may also contribute to these mechanisms.     

A role for PS integrins in morphological growth and synaptic function at the postembryonic neuromuscular junction of Drosophila

A family of three position-specific (PS) integrins are expressed at the Drosophila neuromuscular junction (NMJ): a beta subunit ((betaPS), expressed in both presynaptic and postsynaptic membranes, and two alpha subunits (alphaPS1, alphaPS2), expressed at least in the postsynaptic membrane. PS integrins appear at postembryonic NMJs coincident with the onset of rapid morphological growth and terminal type-specific differentiation, and are restricted to type I synaptic boutons, which mediate fast, excitatory glutamatergic transmission. We show that two distinctive hypomorphic mutant alleles of the beta subunit gene myospheroid (mys(b9) and mys(ts1)), differentially affect betaPS protein expression at the synapse to produce distinctive alterations in NMJ branching, bouton formation, synaptic architecture and the specificity of synapse formation on target cells. The mys(b9) mutation alters betaPS localization to cause a striking reduction in NMJ branching, bouton size/number and the formation of aberrant 'mini-boutons', which may represent a developmentally arrested state. The mys(ts1) mutation strongly reduces betaPS expression to cause the opposite phenotype of excessive synaptic sprouting and morphological growth. NMJ function in these mutant conditions is altered in line with the severity of the morphological aberrations. Consistent with these mutant phenotypes, transgenic overexpression of the betaPS protein with a heat-shock construct or tissue-specific GAL4 drivers causes a reduction in synaptic branching and bouton number. We conclude that betaPS integrin at the postembryonic NMJ is a critical determinant of morphological growth and synaptic specificity. These data provide the first genetic evidence for a functional role of integrins at the postembryonic synapse.     

Moesin contributes an essential structural role in Drosophila photoreceptor morphogenesis

Ezrin-Radixin-Moesin (ERM) family proteins organize heterogeneous sub-plasma membrane protein scaffolds that shape membranes and their physiology. In Drosophila oocytes and imaginal discs, epithelial organization, fundamental to development and physiology, is devastated by the loss of Moesin. Here, we show that Moesin is crucial for Drosophila photoreceptor morphogenesis. Beyond its requirement for retinal epithelium integrity, Moesin is essential for the proper assembly of the apical membrane skeleton that builds the photosensitive membrane, the rhabdomere. Moesin localizes to the rhabdomere base, a dynamic locus of cytoskeletal reorganization and membrane traffic. Downregulation of Moesin through RNAi or genetic loss of function profoundly disrupts the membrane cytoskeleton and apical membrane organization. We find normal levels and distribution of Moesin in photoreceptors of a Moesin mutant previously regarded as protein null, suggesting alternative interpretations for studies using this allele. Our results show an essential structural role for Moesin in photoreceptor morphology.     

Retrograde Gbb signaling through the Bmp type 2 receptor wishful thinking regulates systemic FMRFa expression in Drosophila

Amidated neuropeptides of the FMRFamide class regulate numerous physiological processes including synaptic efficacy at the Drosophila neuromuscular junction (NMJ). We demonstrate here that mutations in wishful thinking (wit) a gene encoding a Drosophila Bmp type 2 receptor that is required for proper neurotransmitter release at the neuromuscular junction, also eliminates expression of FMRFa in that subset of neuroendocrine cells (Tv neurons) which provide the systemic supply of FMRFa peptides. We show that Gbb, a Bmp ligand expressed in the neurohemal organ provides a retrograde signal that helps specify the peptidergic phenotype of the Tv neurons. Finally, we show that supplying FMRFa in neurosecretory cells partially rescues the wit lethal phenotype without rescuing the primary morphological or electrophysiological defects of wit mutants. We propose that Wit and Gbb globally regulate NMJ function by controlling both the growth and transmitter release properties of the synapse as well as the expression of systemic modulators of NMJ synaptic activity.     

Tao Negatively Regulates BMP Signaling During Neuromuscular Junction Development in Drosophila

The coordinated growth and development of synapses is critical for all aspects of neural circuit function and mutations that disrupt these processes can result in various neurological defects. Several anterograde and retrograde signaling pathways, including the canonical Bone Morphogenic Protein (BMP) pathway, regulate synaptic development in vertebrates and invertebrates. At the Drosophila larval neuromuscular junction (NMJ), the retrograde BMP pathway is a part of the machinery that controls NMJ expansion concurrent with larval growth. We sought to determine whether the conserved Hippo pathway, critical for proportional growth in other tissues, also functions in NMJ development. We found that neuronal loss of the serine‐threonine protein kinase Tao, a regulator of the Hippo signaling pathway, results in supernumerary boutons which contain a normal density of active zones. Tao is also required for proper synaptic function, as reduction of Tao results in NMJs with decreased evoked excitatory junctional potentials. Surprisingly, Tao function in NMJ growth is independent of the Hippo pathway. Instead, our experiments suggest that Tao negatively regulates BMP signaling as reduction of Tao leads to an increase in pMad levels in motor neuron nuclei and an increase in BMP target gene expression. Taken together, these results support a role for Tao as a novel inhibitor of BMP signaling in motor neurons during synaptic development and function.     

Drosophila cyfip Regulates Synaptic Development and Endocytosis by Suppressing Filamentous Actin Assembly

The formation of synapses and the proper construction of neural circuits depend on signaling pathways that regulate cytoskeletal structure and dynamics. After the mutual recognition of a growing axon and its target, multiple signaling pathways are activated that regulate cytoskeletal dynamics to determine the morphology and strength of the connection. By analyzing Drosophila mutations in the cytoplasmic FMRP interacting protein Cyfip, we demonstrate that this component of the WAVE complex inhibits the assembly of filamentous actin (F-actin) and thereby regulates key aspects of synaptogenesis. Cyfip regulates the distribution of F-actin filaments in presynaptic neuromuscular junction (NMJ) terminals. At cyfip mutant NMJs, F-actin assembly was accelerated, resulting in shorter NMJs, more numerous satellite boutons, and reduced quantal content. Increased synaptic vesicle size and failure to maintain excitatory junctional potential amplitudes under high-frequency stimulation in cyfip mutants indicated an endocytic defect. cyfip mutants exhibited upregulated bone morphogenetic protein (BMP) signaling, a major growth-promoting pathway known to be attenuated by endocytosis at the Drosophila NMJ. We propose that Cyfip regulates synapse development and endocytosis by inhibiting actin assembly.     

Nervous wreck, an SH3 adaptor protein that interacts with Wsp, regulates synaptic growth in Drosophila

We describe the isolation and characterization of nwk (nervous wreck), a temperature-sensitive paralytic mutant that causes excessive growth of larval neuromuscular junctions (NMJs), resulting in increased synaptic bouton number and branch formation. Ultrastructurally, mutant boutons have reduced size and fewer active zones, associated with a reduction in synaptic transmission. nwk encodes an FCH and SH3 domain-containing adaptor protein that localizes to the periactive zone of presynaptic terminals and binds to the Drosophila ortholog of Wasp (Wsp), a key regulator of actin polymerization. wsp null mutants display synaptic overgrowth similar to nwk and enhance the nwk morphological phenotype in a dose-dependent manner. Evolutionarily, Nwk belongs to a previously undescribed family of adaptor proteins that includes the human srGAPs, which regulate Rho activity downstream of Robo receptors. We propose that Nwk controls synapse morphology by regulating actin dynamics downstream of growth signals in presynaptic terminals.     

Long-term in vitro maintenance of neuromuscular junction activity of Drosophila larvae

The larval Drosophila neuromuscular junction (NMJ) has proven to be an excellent system to test fundamental aspects of synaptic transmission, such as relationships among ion channel function, subtypes of glutamate receptors, and the functions of synaptic proteins in the presynaptic compartment. Recent advances in understanding bi-directional communication between nerves and muscles of Drosophila are helping uncover developmental as well as maintenance cues that could be applicable to all chemical synapses. The development of HL3 medium makes it possible to record synaptic responses at NMJs for prolonged periods of time. We demonstrate that media commonly used to culture CNS neurons and imaginal disks of Drosophila such as Schneider's and M3 completely block glutamatergic synaptic transmission at the NMJ. The depressed postsynaptic excitatory junction potentials (EJPs) partially recover from exposure to such media shortly after switching to the HL3 medium. Preliminary results from NMJs of filleted 3rd instar larvae for 4 days in vitro bathed in a modified HL3 medium show great promise. The resting membrane potential and the EJP amplitudes after 4 days in vitro are normal. These results demonstrate the possibility for chronic studies of developmental regulation in culture, which in some cases are impractical in the whole animal.     

Integrins regulate DLG/FAS2 via a CaM kinase II-dependent pathway to mediate synapse elaboration and stabilization during postembryonic development

Calcium/calmodulin dependent kinase II (CaMKII), PDZ-domain scaffolding protein Discs-large (DLG), immunoglobin superfamily cell adhesion molecule Fasciclin 2 (FAS2) and the position specific (PS) integrin receptors, including betaPS and its alpha partners (alphaPS1, alphaPS2, alphaPS3/alphaVolado), are all known to regulate the postembryonic development of synaptic terminal arborization at the Drosophila neuromuscular junction (NMJ). Recent work has shown that DLG and FAS2 function together to modulate activity-dependent synaptic development and that this role is regulated by activation of CaMKII. We show that PS integrins function upstream of CaMKII in the development of synaptic architecture at the NMJ. betaPS integrin physically associates with the synaptic complex anchored by the DLG scaffolding protein, which contains CaMKII and FAS2. We demonstrate an alteration of the FAS2 molecular cascade in integrin regulatory mutants, as a result of CaMKII/integrin interactions. Regulatory betaPS integrin mutations increase the expression and synaptic localization of FAS2. Synaptic structural defects in betaPS integrin mutants are rescued by transgenic overexpression of CaMKII (proximal in pathway) or genetic reduction of FAS2 (distal in pathway). These studies demonstrate that betaPS integrins act through CaMKII activation to control the localization of synaptic proteins involved in the development of NMJ synaptic morphology.