Visualization of neuropeptide expression, transport, and exocytosis in Drosophila melanogaster.

Neuropeptides affect an extremely diverse set of physiological processes. Neuropeptides are often coreleased with neurotransmitters but, unlike neurotransmitters, the neuropeptide target cells may be distant from the site(s) of secretion. Thus, it is often difficult to measure the amount of neuropeptide release in vivo by electrophysiological methods. Here we establish an in vivo system for studying the developmental expression, processing, transport, and release of neuropeptides. A GFP-tagged atrial natriuretic factor fusion (preproANF-EMD) was expressed in the Drosophila nervous system with the panneural promoter, elav. During embryonic development, proANF-EMD was first seen to accumulate in synaptic regions of the CNS in stage 17 embryos. By the third instar larval stage, highly fluorescent neurons were evident throughout the CNS. In the adult, fluorescence was pronounced in the mushroom bodies, antennal lobe, and the central complex. At the larval neuromuscular junction, proANF-EMD was concentrated in nerve terminals. We compared the release of proANF-EMD from synaptic boutons of NMJ 6/7, which contain almost exclusively glutamate-containing clear vesicles, to those of NMJ 12, which include the peptidergic type III boutons. Upon depolarization, approximately 60% of the tagged neuropeptide was released from NMJs of both muscles in 15 min, as assayed by decreased fluorescence. Although the elav promoter was equally active in the motor neurons that innervate both NMJs 6/7 and 12, NMJ 12 contained 46-fold more neuropeptide and released much more proANF-EMD during stimulation than did NMJ 6/7. Our results suggest that peptidergic neurons have an enhanced ability to accumulate and/or release neuropeptides as compared to neurons that primarily release classical neurotransmitters.     

Neuropeptides and Neuropeptide Receptors: Drug Targets,and Peptide and Non‐Peptide Ligands: a Tribute to Prof. Dieter Seebach

The number of neuropeptides and their corresponding receptors has increased steadily over the last fourty years: initially, peptides were isolated from gut or brain (e.g., Substance P, somatostatin), then by targeted mining in specific regions (e.g., cortistatin, orexin in the brain), or by deorphanization of G‐protein‐coupled receptors (GPCRs; orexin, ghrelin receptors) and through the completion the Human Genome Project. Neuropeptides (and their receptors) have regionally restricted distributions in the central and peripheral nervous system. The neuropeptide signaling is somewhat more distinct spatially than signaling with classical, low‐molecular‐weight neurotransmitters that are more widely expressed, and, therefore, one assumes that drugs acting at neuropeptide receptors may have more selective pharmacological actions with possibly fewer side effects than drugs acting on glutamatergic, GABAergic, monoaminergic, or cholinergic systems. Neuropeptide receptors, which may have a few or multiple subtypes and splice variants, belong almost exclusively to the GPCR family also known as seven‐transmembrane receptors (7TM), a favorite class of drug targets in the pharmaceutical industry. Most neuropeptides are co‐stored and co‐released with classic neurotransmitters, albeit often only at higher frequencies of stimulation or at bursting activity, thus restricting the neuropeptide signaling to specific circumstances, another reason to assume that neuropeptide drug mimics may have less side effects. Neuropeptides possess a wide spectrum of functions from neurohormone, neurotransmitter to growth factor, but also as key inflammatory mediators. Neuropeptides become ‘active’ when the nervous system is challenged, e.g., by stress, injury, drug abuse, or neuropsychiatric disorders with genetic, epigenetic, and/or environmental components. The unsuspected number of true neuropeptides and their cognate receptors provides opportunities to identify novel targets for the treatment of both central and peripheral nervous system disorders. Both, receptor subtype‐selective antagonists and agonists are being developed, as illustrated by the success of somatostatin agonists, angiotensin, and endothelin antagonists, and the expected clinical applications of NK‐1/2/3 (substance P) receptor antagonists, CRF, vasopressin, NPY, neurotensin, orexin antagonists, or neuropeptide receptor modulators; such ligands have efficacy in preclinical or clinical models of pain and neuropsychiatric diseases, such as migraine, chronic/neuropathic pain, anxiety, sleep disorders, depression, and schizophrenia. In addition, both positive and negative allosteric modulators have been described with interesting in vivo activities (e.g., at galanin receptors). The field has become more complex now that an increasing number of heteromeric neuropeptide receptors are described, e.g., ghrelin receptors with 5‐HT_2C or dopamine D₁, D₂ receptors. At long last, structure‐based drug discovery can now be envisaged with confidence, since crystal or solution structure of GPCRs and GPCR? ligand complexes, including peptide receptors, are published almost on a monthly basis. Finally, although most compounds acting at peptide receptors are still peptidomimetics, the last decade has seen the emergence of low‐molecular‐weight nonpeptide ligands (e.g., for orexin, ghrelin, or neurokinin receptors), and surprising progress has been made with β‐ and γ‐peptides as very stable and potent mimetics of, e.g., somatostatin (SRIF), where the native SRIF has a half‐life limited to 2–3 min. This last point will be illustrated more specifically, as we have had a long‐standing collaboration with Prof. D. Seebach to whom this review is dedicated at the occasion of his 75th birthday.     

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.     

Use of targetable gfp‐tagged neuropeptide for visualizing neuropeptide release following execution of a behavior

Previous work has shown that a transgene consisting of a fusion between the rat atrial natriuretic peptide and a green fluorescent protein reporter (ANF‐gfp) is processed, localized, and released, as would be an endogenous neuropeptide when it is expressed in the nervous system of Drosophila melanogaster using the GAL4/UAS expression system. Here we have tested the utility of this targetable transgene for detecting neuropeptide release following the execution of a peptide‐controlled behavior. For the behavior we used ecdysis, the behavior expressed by insects to shed their old cuticle at the end of the molt. We found that larval ecdysis was accompanied by a readily detectable reduction in gfp fluorescence from relevant secretory cells in the periphery and peptidergic neurons in the CNS. We also found that expression of the ANF‐gfp products did not have detrimental effects on larval ecdysis or adult circadian rhythmicity, when the transgene was expressed in peptidergic cells that are known to control these behaviors. Finally, we used a broadly expressed GAL4 driver to show that the UAS‐ANF‐gfp transgene could be used to identify axons that show a reduction in gfp fluorescence following the expression of ecdysis behavior. These findings, coupled with the availability of an increasing number of strains bearing different GAL4 drivers, suggest that this transgene will be a useful tool for identifying peptidergic neurons and secretory cells (and, eventually, their secretory product) that release their peptide content during the occurrence, in the intact animal, of a developmental, physiological or behavioral process of interest. © 2004 Wiley Periodicals, Inc. J Neurobiol 59: 181–191, 2004     

Identification of a complex peptidergic neuroendocrine network in the hard tick, <Emphasis Type="Italic">Rhipicephalus appendiculatus</Emphasis>

Neuropeptides are crucial regulators of development and various physiological functions but little is known about their identity, expression and function in vectors of pathogens causing serious diseases, such as ticks. Therefore, we have used antibodies against multiple insect and crustacean neuropeptides to reveal the presence of these bioactive molecules in peptidergic neurons and cells of the ixodid tick Rhipicephalus appendiculatus. These antibodies have detected 15 different immunoreactive compounds expressed in specific central and peripheral neurons associated with the synganglion. Most central neurons arborize in distinct areas of the neuropile or the putative neurohaemal periganglionic sheath of the synganglion. Several large identified neurons in the synganglion project multiple processes through peripheral nerves to form elaborate axonal arborizations on the surface of salivary glands or to terminate in the lateral segmental organs (LSO). Additional neuropeptide immunoreactivity has been observed in intrinsic secretory cells of the LSO. We have also identified two novel clusters of peripheral neurons embedded in the cheliceral and paraspiracular nerves. These neurons project branching axons into the synganglion and into the periphery. Our study has thus revealed a complex network of central and peripheral peptidergic neurons, putative neurohaemal and neuromodulatory structures and endocrine cells in the tick comparable with those found in insect and crustacean neuroendocrine systems. Strong specific staining with a large variety of antibodies also indicates that the tick nervous system and adjacent secretory organs are rich sources of diverse neuropeptides related to those identified in insects, crustaceans or even vertebrates. This work was supported by Slovak grant agencies: Agentúra na podporu vyskumu a vyvoja (APVV-51-039105) and Vedecká grantová agentúra (VEGA 2-6090-26 and 2/6155/26).     

A Highlights from MBoC Selection: Synaptic neuropeptide release by dynamin-dependent partial release from circulating vesicles

Neurons release neuropeptides, enzymes, and neurotrophins by exocytosis of dense-core vesicles (DCVs). Peptide release from individual DCVs has been imaged in vitro with endocrine cells and at the neuron soma, growth cones, neurites, axons, and dendrites but not at nerve terminals, where peptidergic neurotransmission occurs. Single presynaptic DCVs have, however, been tracked in native terminals with simultaneous photobleaching and imaging (SPAIM) to show that DCVs undergo anterograde and retrograde capture as they circulate through en passant boutons. Here dynamin (encoded by the shibire gene) is shown to enhance activity-evoked peptide release at the Drosophila neuromuscular junction. SPAIM demonstrates that activity depletes only a portion of a single presynaptic DCV''s content. Activity initiates exocytosis within seconds, but subsequent release occurs slowly. Synaptic neuropeptide release is further sustained by DCVs undergoing multiple rounds of exocytosis. Synaptic neuropeptide release is surprisingly similar regardless of anterograde or retrograde DCV transport into boutons, bouton location, and time of arrival in the terminal. Thus vesicle circulation and bidirectional capture supply synapses with functionally competent DCVs. These results show that activity-evoked synaptic neuropeptide release is independent of a DCV''s past traffic and occurs by slow, dynamin-dependent partial emptying of DCVs, suggestive of kiss-and-run exocytosis.     

The adult abdominal neuromuscular junction of Drosophila: a model for synaptic plasticity

During its life cycle, Drosophila makes two sets of neuromuscular junctions (NMJs), embryonic/larval and adult, which serve distinct stage-specific functions. During metamorphosis, the larval NMJs are restructured to give rise to their adult counterparts, a process that is integrated into the overall remodeling of the nervous system. The NMJs of the prothoracic muscles and the mesothoracic dorsal longitudinal (flight) muscles have been previously described. Given the diversity and complexity of adult muscle groups, we set out to examine the less complex abdominal muscles. The large bouton sizes of these NMJs are particularly advantageous for easy visualization. Specifically, we have characterized morphological attributes of the ventral abdominal NMJ and show that an embryonic motor neuron identity gene, dHb9, is expressed at these adult junctions. We quantified bouton numbers and size and examined the localization of synaptic markers. We have also examined the formation of boutons during metamorphosis and examined the localization of presynaptic markers at these stages. To test the usefulness of the ventral abdominal NMJs as a model system, we characterized the effects of altering electrical activity and the levels of the cell adhesion molecule, FasciclinII (FasII). We show that both manipulations affect NMJ formation and that the effects are specific as they can be rescued genetically. Our results indicate that both activity and FasII affect development at the adult abdominal NMJ in ways that are distinct from their larval and adult thoracic counterparts     

Neurotransmitters and neuropeptides in the developing human central nervous system. A review

This is a review on the ontogenesis of major neurotransmitters and neuropeptides in the developing human central nervous system. In general, the molecules under study appeared early in development, usually in the first trimester. Cholinergic neurons were found to be present around the time of neuropeptide formation. The newly formed neuropeptidergic fibers extended towards the cholinergic centers where both might interact. In the major centers of the central nervous system, neuropeptides were also noted to colocalize with various neurotransmitters. For example, in the facial nucleus, enkepahlin and substance P fibers coexisted with cholinergic and catecholaminergic neurons, suggesting complex interactions. In the interpeduncular nucleus, peptidergic neurons acting as interneurons clearly modulated the afferent input to this nucleus. In the hippocampus and in sensory organs such as the retina, there were indications that neuropeptides and gamma-amino butyric acid coexisted. We hypothesize that interactions of neurotransmitters and peptides in neurons and fibers early in development play an indispensable role in the morphogenesis of the human central nervous system.     

Jelly belly trans‐synaptic signaling to anaplastic lymphoma kinase regulates neurotransmission strength and synapse architecture

In Drosophila, the secreted signaling molecule Jelly Belly (Jeb) activates anaplastic lymphoma kinase (Alk), a receptor tyrosine kinase, in multiple developmental and adult contexts. We have shown previously that Jeb and Alk are highly enriched at Drosophila synapses within the CNS neuropil and neuromuscular junction (NMJ) and postulated a conserved intercellular signaling function. At the embryonic and larval NMJ, Jeb is localized in the motor neuron presynaptic terminal whereas Alk is concentrated in the muscle postsynaptic domain surrounding boutons, consistent with anterograde trans‐synaptic signaling. Here, we show that neurotransmission is regulated by Jeb secretion by functional inhibition of Jeb–Alk signaling. Jeb is a novel negative regulator of neuromuscular transmission. Reduction or inhibition of Alk function results in enhanced synaptic transmission. Activation of Alk conversely inhibits synaptic transmission. Restoration of wild‐type postsynaptic Alk expression in Alk partial loss‐of‐function mutants rescues NMJ transmission phenotypes and confirms that postsynaptic Alk regulates NMJ transmission. The effects of impaired Alk signaling on neurotransmission are observed in the absence of associated changes in NMJ structure. Complete removal of Jeb in motor neurons, however, disrupts both presynaptic bouton architecture and postsynaptic differentiation. Nonphysiologic activation of Alk signaling also negatively regulates NMJ growth. Activation of Jeb–Alk signaling triggers the Ras‐MAP kinase cascade in both pre‐ and postsynaptic compartments. These novel roles for Jeb–Alk signaling in the modulation of synaptic function and structure have potential implications for recently reported Alk functions in human addiction, retention of spatial memory, cognitive dysfunction in neurofibromatosis, and pathogenesis of amyotrophic lateral sclerosis. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Distribution of neuropeptides in the antennal lobes of male Spodoptera littoralis

Olfaction is an important sensory modality that regulates a plethora of behavioural expressions in insects. Processing of olfactory information takes place in the primary olfactory centres of the brain, namely the antennal lobes (ALs). Neuropeptides have been shown to be present in the olfactory system of various insect species. In the present study, we analyse the distribution of tachykinin, FMRFamide-related peptides, allatotropin, allatostatin, myoinhibitory peptides and SIFamide in the AL of the male Egyptian cotton leafworm, Spodoptera littoralis. Immunocytochemical analyses revealed that most neuropeptides were expressed in different subpopulations of AL neurons. Their arborisation patterns within the AL suggest a significant role of neuropeptide signalling in the modulation of AL processing. In addition to local interneurons, our analysis also revealed a diversity of extrinsic peptidergic neurons that connected the antennal lobe with other brain centres. Their distributions suggest that extrinsic neurons perform various types of context-related modulation.     

Neuropeptide signaling near and far: how localized and timed is the action of neuropeptides in brain circuits?

Neuropeptide signaling is functionally very diverse and one and the same neuropeptide may act as a circulating neurohormone, as a locally released neuromodulator or even as a cotransmitter of classical fast-acting neurotransmitters. Thus, neuropeptides are produced by a huge variety of neuron types in different parts of the nervous system. Within the central nervous system (CNS) there are numerous types of peptidergic interneurons, some with strictly localized and patterned branching morphologies, others with widespread and diffuse arborizations. From morphology alone it is often difficult to predict the sphere of influence of a peptidergic interneuron, especially since it has been shown that neuropeptides can diffuse over tens of micrometers within neuropils, and that peptides probably are released exclusively in perisynaptic (or non-synaptic) regions. This review addresses some questions related to peptidergic signaling in the insect CNS. How diverse are the spatial relations between peptidergic neurons and their target neurons and what determines the sphere of functional influence? At one extreme there is volume transmission and at the other targeted cotransmission at synapses. Also temporal aspects of peptidergic signaling are of interest: how transient are peptidergic messages? Factors important for these spatial and temporal aspects of peptidergic signaling are proximity between release sites and cognate receptors, distribution of peptidase activity that can terminate peptide action and colocalization of other neuroactive compounds in the presynaptic peptidergic neuron (and corresponding receptors in target neurons). Other factors such as expression of different channel types, receptor inactivation mechanisms and second messenger systems probably also contribute to the diversity in temporal properties of peptide signaling.     

Hypothalamic Sirt1 protects terminal Schwann cells and neuromuscular junctions from age‐related morphological changes

Neuromuscular decline occurs with aging. The neuromuscular junction (NMJ), the interface between motor nerve and muscle, also undergoes age‐related changes. Aging effects on the NMJ components—motor nerve terminal, acetylcholine receptors (AChRs), and nonmyelinating terminal Schwann cells (tSCs)—have not been comprehensively evaluated. Sirtuins delay mammalian aging and increase longevity. Increased hypothalamic Sirt1 expression results in more youthful physiology, but the relationship between NMJ morphology and hypothalamic Sirt1 was previously unknown. In wild‐type mice, all NMJ components showed age‐associated morphological changes with ~80% of NMJs displaying abnormalities by 17 months of age. Aged mice with brain‐specific Sirt1 overexpression (BRASTO) had more youthful NMJ morphologic features compared to controls with increased tSC numbers, increased NMJ innervation, and increased numbers of normal AChRs. Sympathetic NMJ innervation was increased in BRASTO mice. In contrast, hypothalamic‐specific Sirt1 knockdown led to tSC abnormalities, decreased tSC numbers, and more denervated endplates compared to controls. Our data suggest that hypothalamic Sirt1 functions to protect NMJs in skeletal muscle from age‐related changes via sympathetic innervation.     

Current and emerging methods for probing neuropeptide transmission

Neuropeptides comprise the most diverse category of neurochemicals in the brain, playing critical roles in a wide range of physiological and pathophysiological processes. Monitoring neuropeptides with high spatial and temporal resolution is essential for understanding how peptidergic transmission is regulated throughout the central nervous system. In this review, we provide an overview of current non-optical and optical approaches used to detect neuropeptides, including their design principles, intrinsic properties, and potential limitations. We also highlight the advantages of using G protein‒coupled receptor (GPCR) activation‒based (GRAB) sensors to monitor neuropeptides in vivo with high sensitivity, good specificity, and high spatiotemporal resolution. Finally, we present a promising outlook regarding the development and optimization of new GRAB neuropeptide sensors, as well as their potential applications.     

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.     

Regulation of peptidergic vesicle mobility by secretagogues

Neuropeptides are released into the extracellular space from large secretory granules. In order to reach their release sites, these granules are translocated on microtubules and thought to interact with filamentous actin as they approach the cell membrane. We have used a green fluorescent protein-tagged neuropeptide prohormone (prepro-orphanin FQ) to visualize vesicle trafficking dynamics in NS20Y cells and cultures of primary hippocampal neurons. We found that the majority of secretory granules were mobile and accumulated at both the tips of neurites as well as other apparently specialized cellular sites. We also used live-cell imaging to test the notion that peptidergic vesicle mobility was regulated by secretagogues. We show that treatment with forskolin appeared to increase vesicle rates of speed, while depolarization with high K⁺ had no effect, even though both treatments stimulated neuropeptide secretion. In cultured hippocampal neurons the green fluorescent protein-tagged secretory vesicles were routed to both dendrites and axons, indicating that peptidergic vesicle transport was not polarized. Basal peptidergic vesicle mobility rates in hippocampal neurons were the same as those in NS20Y cells. Taken together, these studies suggest that secretory vesicle mobility is regulated by specific classes of secretagogues and that neuropeptide containing secretory vesicles may be released from dendritic structures.     

Cell interactions that determine sympathetic neuron transmitter phenotype and the neurokines that mediate them

The transmitter properties of both developing and mature sympathetic neurons are plastic and can be modulated by a number of environmental cues. Cell culture studies demonstrate that noradrenergic neurons can be induced to become cholinergic and that the expression of neuropeptides can be altered. Similar changes in transmitter phenotype occur in vivo. During development, noradrenergic neurons that innervate eccrine sweat glands acquire cholinergic and peptidergic function. This change is dependent upon interactions with the target tissue. Following injury of sympathetic neurons in developing and adult animals, striking alterations take place in peptide expression. Ciliary neurotrophic factor and cholinergic differentiation factor/leukemia inhibitory factor, members of a family that includes several hematopoeitic cytokines, induce cholinergic function and modulate neuropeptide expression in cultured sympathetic neurons. Studies in progress provide evidence that members of this new cytokine family influence the transmitter phenotype of sympathetic neurons not only in vitro but also in vivo. © 1993 John Wiley & Sons, Inc.     

Drosophila adducin regulates Dlg phosphorylation and targeting of Dlg to the synapse and epithelial membrane

Adducin is a cytoskeletal protein having regulatory roles that involve actin filaments, functions that are inhibited by phosphorylation of adducin by protein kinase C. Adducin is hyperphosphorylated in nervous system tissue in patients with the neurodegenerative disease amyotrophic lateral sclerosis, and mice lacking β-adducin have impaired synaptic plasticity and learning. We have found that Drosophila adducin, encoded by hu-li tai shao (hts), is localized to the post-synaptic larval neuromuscular junction (NMJ) in a complex with the scaffolding protein Discs large (Dlg), a regulator of synaptic plasticity during growth of the NMJ. hts mutant NMJs are underdeveloped, whereas over-expression of Hts promotes Dlg phosphorylation, delocalizes Dlg away from the NMJ, and causes NMJ overgrowth. Dlg is a component of septate junctions at the lateral membrane of epithelial cells, and we show that Hts regulates Dlg localization in the amnioserosa, an embryonic epithelium, and that embryos doubly mutant for hts and dlg exhibit defects in epithelial morphogenesis. The phosphorylation of Dlg by the kinases PAR-1 and CaMKII has been shown to disrupt Dlg targeting to the NMJ and we present evidence that Hts regulates Dlg targeting to the NMJ in muscle and the lateral membrane of epithelial cells by controlling the protein levels of PAR-1 and CaMKII, and consequently the extent of Dlg phosphorylation.     

The PDZ-GEF Gef26 regulates synapse development and function via FasII and Rap1 at the Drosophila neuromuscular junction

Guanine nucleotide exchange factors (GEFs) are essential for small G proteins to activate their downstream signaling pathways, which are involved in morphogenesis, cell adhesion, and migration. Mutants of Gef26, a PDZ-GEF (PDZ domain-containing guanine nucleotide exchange factor) in Drosophila, exhibit strong defects in wings, eyes, and the reproductive and nervous systems. However, the precise roles of Gef26 in development remain unclear. In the present study, we analyzed the role of Gef26 in synaptic development and function. We identified significant decreases in bouton number and branch length at larval neuromuscular junctions (NMJs) in Gef26 mutants, and these defects were fully rescued by restoring Gef26 expression, indicating that Gef26 plays an important role in NMJ morphogenesis. In addition to the observed defects in NMJ morphology, electrophysiological analyses revealed functional defects at NMJs, and locomotor deficiency appeared in Gef26 mutant larvae. Furthermore, Gef26 regulated NMJ morphogenesis by regulating the level of synaptic Fasciclin II (FasII), a well-studied cell adhesion molecule that functions in NMJ development and remodeling. Finally, our data demonstrate that Gef26-specific small G protein Rap1 worked downstream of Gef26 to regulate the level of FasII at NMJs, possibly through a βPS integrin-mediated signaling pathway. Taken together, our findings define a novel role of Gef26 in regulating NMJ development and function.