Drosophila Nervous System Research Articles

Intrinsic dorsoventral patterning and extrinsic EGFR signaling genes control glial cell development in the Drosophila nervous system

Dorsoventral patterning and epidermal growth factor receptor (EGFR) signaling genes are essential for determining neural identity and differentiation of the Drosophila nervous system. Their role in glial cell development in the Drosophila nervous system is not clearly established. Our study demonstrated that the dorsoventral patterning genes, vnd, ind, and msh, are intrinsically essential for the proper expression of a master glial cell regulator, gcm, and a differentiation gene, repo, in the lateral glia. In addition, we showed that esg is particularly required for their expression in the peripheral glia. These results indicate that the dorsoventral patterning and EGFR signaling genes are essential for identity determination and differentiation of the lateral glia by regulating proper expression of gcm and repo in the lateral glia from the early glial development. In contrast, overexpression of vnd, msh, spi, and Egfr genes repressed the expression of Repo in the ventral neuroectoderm, indicating that maintenance of correct columnar identity along the dorsoventral axis by proper expression of these genes is essential for restrictive formation of glial precursor cells in the lateral neuroectoderm. Therefore, the dorsoventral patterning and EGFR signaling genes play essential roles in correct identity determination and differentiation of lateral glia in the Drosophila nervous system.

Long term ex vivo culturing of Drosophila brain as a method to live image pupal brains: insights into the cellular mechanisms of neuronal remodeling.

Holometabolous insects, including Drosophila melanogaster, undergo complete metamorphosis that includes a pupal stage. During metamorphosis, the Drosophila nervous system undergoes massive remodeling and growth, that include cell death and large-scale axon and synapse elimination as well as neurogenesis, developmental axon regrowth, and formation of new connections. Neuronal remodeling is an essential step in the development of vertebrate and invertebrate nervous systems. Research on the stereotypic remodeling of Drosophila mushroom body (MB) γ neurons has contributed to our knowledge of the molecular mechanisms of remodeling but our knowledge of the cellular mechanisms remain poorly understood. A major hurdle in understanding various dynamic processes that occur during metamorphosis is the lack of time-lapse resolution. The pupal case and opaque fat bodies that enwrap the central nervous system (CNS) make live-imaging of the central brain in-vivo impossible. We have established an ex vivo long-term brain culture system that supports the development and neuronal remodeling of pupal brains. By optimizing culture conditions and dissection protocols, we have observed development in culture at kinetics similar to what occurs in vivo. Using this new method, we have obtained the first time-lapse sequence of MB γ neurons undergoing remodeling in up to a single cell resolution. We found that axon pruning is initiated by blebbing, followed by one-two nicks that seem to initiate a more widely spread axon fragmentation. As such, we have set up some of the tools and methodologies needed for further exploration of the cellular mechanisms of neuronal remodeling, not limited to the MB. The long-term ex vivo brain culture system that we report here could be used to study dynamic aspects of neurodevelopment of any Drosophila neuron.

Differentiation of Drosophila glial cells.

Glial cells are important constituents of the nervous system and a hallmark of these cells are their pronounced migratory abilities. In Drosophila, glial lineages have been well described and some of the molecular mechanisms necessary to guide migrating glial cells to their final target sites have been identified. With the onset of migration, glial cells are already specified into one of five main glial cell types. The perineurial and subperineurial glial cells are eventually located at the outer surface of the Drosophila nervous system and constitute the blood-brain barrier. The cortex glial cells ensheath all neuroblasts and their progeny and reside within the central nervous system. Astrocyte-like cells invade the neuropil to control synaptic function and ensheathing glial cells encase the entire neuropil. Within the peripheral nervous system, wrapping glial cells ensheath individual axons or axon fascicles. Here, we summarize the current knowledge on how differentiation of glial cells into the specific subtypes is orchestrated. Furthermore, we discuss sequencing data that will facilitate further analyses of glial differentiation in the fly nervous system.

The Organization of Mitochondrial Quality Control and Life Cycle in the Nervous System In Vivo in the Absence of PINK1.

Maintenance of healthy mitochondria is crucial in cells, such as neurons, with high metabolic demands, and dysfunctional mitochondria are thought to be selectively degraded. Studies of chemically uncoupled cells have implicated PINK1 mitochondrial kinase, and Parkin E3 ubiquitin ligase in targeting depolarized mitochondria for degradation. However, the role of the PINK1/Parkin pathway in mitochondrial turnover is unclear in the nervous system under normal physiological conditions, and we understand little about the changes that occur in the mitochondrial life cycle when turnover is disrupted. Here, we evaluated the nature, location, and regulation of quality control in vivo using quantitative measurements of mitochondria in Drosophila nervous system, with deletion and overexpression of genes in the PINK1/Parkin pathway. We tested the hypotheses that impairment of mitochondrial quality control via suppression of PINK1 function should produce failures of turnover, accumulation of senescent mitochondria in the axon, defects in mitochondrial traffic, and a significant shift in the mitochondrial fission-fusion steady state. Although mitochondrial membrane potential was diminished by PINK1 deletion, we did not observe the predicted increases in mitochondrial density or length in axons. Loss of PINK1 also produced specific, directionally balanced defects in mitochondrial transport, without altering the balance between stationary and moving mitochondria. Somatic mitochondrial morphology was also compromised. These results strongly circumscribe the possible mechanisms of PINK1 action in the mitochondrial life cycle and also raise the possibility that mitochondrial turnover events that occur in cultured embryonic axons might be restricted to the cell body in vivo, in the intact nervous system.

Drosophila nervous system as a target of aging and anti-aging interventions.

Nervoussystemregulateshomeostasisandadaptationtoenvironmentalchangesofawholeorgan-ism, thus deregulation of nervous processes accelerates aging (Alcedo et al., 2013a,b). The agingprocess in different models is associated with progressive degeneration of the nervous system (Leeet al., 2000) and progression of age-related neurodegenerative diseases such as Alzheimer’s dis-ease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis (Boerrigter et al.,1992; Coppede and Migliore, 2010). The neurodegeneration also characterizes the progeroid syn-dromes,includingHutchinson-GilfordsyndromeandWerner’ssyndrome(CoppedeandMigliore,2010).Drosophila melanogaster is a good model organism to study age-related neurodegenerativechanges (Lu and Vogel, 2009). Enrichment in mutants with neurodegeneration among flies withshortened lifespan has been reported (Buchanan and Benzer, 1993; Kretzschmar et al., 1997). Thebrain from old flies demonstrates the ultrastructural neurodegenerative changes such as reductionin the number of synapses, defects in mitochondria, and increase in neuronal apoptosis (Had-dadietal.,2014).However,anti-aginginterventionsmaypostponetheneurodegeneration(Bgatovaet al., 2015).Here we consider molecular genetic changes in the Drosophilaaging brain and the bases forapplying the brain as a target for anti-aging intervention.

The Drosophila blood-brain barrier: development and function of a glial endothelium.

The efficacy of neuronal function requires a well-balanced extracellular ion homeostasis and a steady supply with nutrients and metabolites. Therefore, all organisms equipped with a complex nervous system developed a so-called blood-brain barrier, protecting it from an uncontrolled entry of solutes, metabolites or pathogens. In higher vertebrates, this diffusion barrier is established by polarized endothelial cells that form extensive tight junctions, whereas in lower vertebrates and invertebrates the blood-brain barrier is exclusively formed by glial cells. Here, we review the development and function of the glial blood-brain barrier of Drosophila melanogaster. In the Drosophila nervous system, at least seven morphologically distinct glial cell classes can be distinguished. Two of these glial classes form the blood-brain barrier. Perineurial glial cells participate in nutrient uptake and establish a first diffusion barrier. The subperineurial glial (SPG) cells form septate junctions, which block paracellular diffusion and thus seal the nervous system from the hemolymph. We summarize the molecular basis of septate junction formation and address the different transport systems expressed by the blood-brain barrier forming glial cells.

Lack of evidence of the interaction of the Aβ peptide with the Wnt signaling cascade in Drosophila models of Alzheimer's disease.

BackgroundAlzheimer’s disease (AD) is the leading form of dementia worldwide. The Aβ-peptide is believed to be the major pathogenic compound of the disease. Since several years it is hypothesized that Aβ impacts the Wnt signaling cascade and therefore activation of this signaling pathway is proposed to rescue the neurotoxic effect of Aβ.FindingsExpression of the human Aβ42 in the Drosophila nervous system leads to a drastically shortened life span. We found that the action of Aβ42 specifically in the glutamatergic motoneurons is responsible for the reduced survival. However, we find that the morphology of the glutamatergic larval neuromuscular junctions, which are widely used as the model for mammalian central nervous system synapses, is not affected by Aβ42 expression. We furthermore demonstrate that genetic activation of the Wnt signal transduction pathway in the nervous system is not able to rescue the shortened life span or a rough eye phenotype in Drosophila.ConclusionsOur data confirm that the life span is a useful readout of Aβ42 induced neurotoxicity in Drosophila; the neuromuscular junction seems however not to be an appropriate model to study AD in flies. Additionally, our results challenge the hypothesis that Wnt signaling might be implicated in Aβ42 toxicity and might serve as a drug target against AD.

Single Serotonergic Neurons that Modulate Aggression in Drosophila

Monoamine serotonin (5HT) has been linked to aggression for many years across species. However, elaboration of the neurochemical pathways that govern aggression has proven difficult because monoaminergic neurons also regulate other behaviors. There are approximately 100 serotonergic neurons in the Drosophila nervous system, and they influence sleep, circadian rhythms, memory, and courtship. In the Drosophila model of aggression, the acute shut down of the entire serotonergic system yields flies that fight less, whereas induced activation of 5HT neurons promotes aggression. Using intersectional genetics, we restricted the population of 5HT neurons that can be reproducibly manipulated to identify those that modulate aggression. Although similar approaches were used recently to find aggression-modulating dopaminergic and Fru(M)-positive peptidergic neurons, the downstream anatomical targets of the neurons that make up aggression-controlling circuits remain poorly understood. Here, we identified a symmetrical pair of serotonergic PLP neurons that are necessary for the proper escalation of aggression. Silencing these neurons reduced aggression in male flies, and activating them increased aggression in male flies. GFP reconstitution across synaptic partners (GRASP) analyses suggest that 5HT-PLP neurons form contacts with 5HT1A receptor-expressing neurons in two distinct anatomical regions of the brain. Activation of these 5HT1A receptor-expressing neurons, in turn, caused reductions in aggression. Our studies, therefore, suggest that aggression may be held in check, at least in part, by inhibitory input from 5HT1A receptor-bearing neurons, which can be released by activation of the 5HT-PLP neurons.

Glia in Drosophila behavior.

Glial cells constitute about 10% of the Drosophila nervous system. The development of genetic and molecular tools has helped greatly in defining different types of glia. Furthermore, considerable progress has been made in unraveling the mechanisms that control the development and differentiation of Drosophila glia. By contrast, the role of glia in adult Drosophila behavior is not well understood. We here summarize recent work describing the role of glia in normal behavior and in Drosophila models for neurological and behavioral disorders.

A study of Drosophila spinster expression and its functions during embryogenesis

During the development of the Drosophila nervous system, the programmed cell death (PCD) regulates the cell number. The spinster (spin) encodes a multiple transmembrane protein and females showed a strong rejection response against the courting males. Mutation in spin interferes with the PCD of neurons, which subsequently induce the degeneration of adult neural cells. However, this spin functions has not been investigated yet during embryogenesis. In this study we first examined spin expression in detail and its function during embryonic development. Spin was primarily expressed in surface glial cells, including subperineurial glial cells and exit glia, but not in neural cells. In spin loss-of-function mutant embryos, Glial cells increased in number, and neural overgrowth occurred. In spin gain-of-function mutant embryos, PNS was predominantly degenerated at late embryonic stages. These results indicate that spin is involved in neurogenesis via cell death during embryogenesis.

AMPK Modulates Tissue and Organismal Aging in a Non-Cell-Autonomous Manner

AMPK exerts prolongevity effects in diverse species; however, the tissue-specific mechanisms involved are poorly understood. Here, we show that upregulation of AMPK in the adult Drosophila nervous system induces autophagy both in the brain and also in the intestinal epithelium. Induction of autophagy is linked to improved intestinal homeostasis during aging and extended lifespan. Neuronal upregulation of the autophagy-specific protein kinase Atg1 is both necessary and sufficient to induce these intertissue effects during aging and to prolong the lifespan. Furthermore, upregulation of AMPK in the adult intestine induces autophagy both cell autonomously and non-cell-autonomously in the brain, slows systemic aging, and prolongs the lifespan. We show that the organism-wide response to tissue-specific AMPK/Atg1 activation is linked to reduced insulin-like peptide levels in the brain and a systemic increase in 4E-BP expression. Together, these results reveal that localized activation of AMPK and/or Atg1 in key tissues can slow aging in a non-cell-autonomous manner.

Drosophila adult olfactory shock learning.

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the elucidation of the molecular mechanisms involved in cognitive diseases1-7. Learning and memory can be assayed in larvae to study the effect of neurodevelopmental genes8-10 and in flies to measure the contribution of adult plasticity genes1-7. Furthermore, the short lifespan of Drosophila facilitates the analysis of genes mediating age-related memory impairment5,11-13. The availability of many inducible promoters that subdivide the Drosophila nervous system makes it possible to determine when and where a gene of interest is required for normal memory as well as relay of different aspects of the reinforcement signal3,4,14,16.Studying memory in adult Drosophila allows for a detailed analysis of the behavior and circuitry involved and a measurement of long-term memory15-17. The length of the adult stage accommodates longer-term genetic, behavioral, dietary and pharmacological manipulations of memory, in addition to determining the effect of aging and neurodegenerative disease on memory3-6,11-13,15-21.Classical conditioning is induced by the simultaneous presentation of a neutral odor cue (conditioned stimulus, CS+) and a reinforcement stimulus, e.g., an electric shock or sucrose, (unconditioned stimulus, US), that become associated with one another by the animal1,16. A second conditioned stimulus (CS-) is subsequently presented without the US. During the testing phase, Drosophila are simultaneously presented with CS+ and CS- odors. After the Drosophila are provided time to choose between the odors, the distribution of the animals is recorded. This procedure allows associative aversive or appetitive conditioning to be reliably measured without a bias introduced by the innate preference for either of the conditioned stimuli. Various control experiments are also performed to test whether all genotypes respond normally to odor and reinforcement alone.

<em>Drosophila</em> Adult Olfactory Shock Learning

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the elucidation of the molecular mechanisms involved in cognitive diseases1-7. Learning and memory can be assayed in larvae to study the effect of neurodevelopmental genes8-10 and in flies to measure the contribution of adult plasticity genes1-7. Furthermore, the short lifespan of Drosophila facilitates the analysis of genes mediating age-related memory impairment5,11-13. The availability of many inducible promoters that subdivide the Drosophila nervous system makes it possible to determine when and where a gene of interest is required for normal memory as well as relay of different aspects of the reinforcement signal3,4,14,16. Studying memory in adult Drosophila allows for a detailed analysis of the behavior and circuitry involved and a measurement of long-term memory15-17. The length of the adult stage accommodates longer-term genetic, behavioral, dietary and pharmacological manipulations of memory, in addition to determining the effect of aging and neurodegenerative disease on memory3-6,11-13,15-21. Classical conditioning is induced by the simultaneous presentation of a neutral odor cue (conditioned stimulus, CS+) and a reinforcement stimulus, e.g., an electric shock or sucrose, (unconditioned stimulus, US), that become associated with one another by the animal1,16. A second conditioned stimulus (CS-) is subsequently presented without the US. During the testing phase, Drosophila are simultaneously presented with CS+ and CS- odors. After the Drosophila are provided time to choose between the odors, the distribution of the animals is recorded. This procedure allows associative aversive or appetitive conditioning to be reliably measured without a bias introduced by the innate preference for either of the conditioned stimuli. Various control experiments are also performed to test whether all genotypes respond normally to odor and reinforcement alone.

Closing the Gap between Glia and Neuroblast Proliferation

Reporting in this issue of Developmental Cell, Spéder and Brand (2014) show that gap junctions are required in blood-brain barrier glial cells to reactivate proliferation of quiescent neuroblasts. Gap junctions allow synchronous Ca(2+) waves and control insulin-like protein Dipl6 expression and secretion to trigger neuroblast division.

Interlocked loops trigger lineage specification and stable fates in the Drosophila nervous system.

Multipotent precursors are plastic cells that generate different, stable fates at the correct number, place and time, to allow tissue and organ formation. While fate determinants are known to trigger specific transcriptional programs, the molecular pathway driving the progression from multipotent precursors towards stable and specific identities remains poorly understood. Here we demonstrate that, in Drosophila neural precursors, the glial determinant glial cell missing (Gcm) acts as a 'time bomb' and triggers its own degradation once the glial programme is stably activated. This requires a sequence of transcriptional and posttranscriptional loops, whereby a Gcm target first affects the expression and then acetylation of the fate determinant, thus controlling Gcm levels and stability over time. Defective homeostasis between the loops alters the neuron:glia ratio and freezes cells in an intermediate glial/neuronal phenotype. In sum, we identify an efficient strategy triggering cell identity, a process altered in pathological conditions such as cancer.

Glia ECM interactions are required to shape the Drosophila nervous system

Organs are characterized by a specific shape that is often remodeled during development. The dynamics of organ shape is in particular evident during the formation of the Drosophila nervous system. During embryonic stages the central nervous system compacts, whereas selective growth occurs during larval stages. The nervous system is covered by a layer of surface glial cells that form the blood brain barrier and a thick extracellular matrix called neural lamella. The size of the neural lamella is dynamically adjusted to the growing nervous system and we show here that perineurial glial cells secrete proteases to remodel this matrix. Moreover, an imbalance in proteolytic activity results in an abnormal shape of the nervous system. To identify further components controlling nervous system shape we performed an RNAi based screen and identified the gene nolo, which encodes an ADAMTS-like protein. We generated loss of function alleles and demonstrate a requirement in glial cells. Mutant nolo larvae, however, do not show an abnormal nervous system shape. The only predicted off-target of the nolodsRNA is Oatp30B, which encodes an organic anion transporting protein characterized by an extracellular protease inhibitor domain. Loss of function mutants were generated and double mutant analyses demonstrate a genetic interaction between nolo and Oatp30B which prevented the generation of maternal zygotic mutant larvae.

Terminal axonal arborization and synaptic bouton formation critically rely on abp1 and the arp2/3 complex.

Neuronal network formation depends on properly timed and localized generation of presynaptic as well as postsynaptic structures. Although of utmost importance for understanding development and plasticity of the nervous system and neurodegenerative diseases, the molecular mechanisms that ensure the fine-control needed for coordinated establishment of pre- and postsynapses are still largely unknown. We show that the F-actin-binding protein Abp1 is prominently expressed in the Drosophila nervous system and reveal that Abp1 is an important regulator in shaping glutamatergic neuromuscular junctions (NMJs) of flies. STED microscopy shows that Abp1 accumulations can be found in close proximity of synaptic vesicles and at the cell cortex in nerve terminals. Abp1 knock-out larvae have locomotion defects and underdeveloped NMJs that are characterized by a reduced number of both type Ib synaptic boutons and branches of motornerve terminals. Abp1 is able to indirectly trigger Arp2/3 complex-mediated actin nucleation and interacts with both WASP and Scar. Consistently, Arp2 and Arp3 loss-of-function also resulted in impairments of bouton formation and arborization at NMJs, i.e. fully phenocopied abp1 knock-out. Interestingly, neuron- and muscle-specific rescue experiments revealed that synaptic bouton formation critically depends on presynaptic Abp1, whereas the NMJ branching defects can be compensated for by restoring Abp1 functions at either side. In line with this presynaptic importance of Abp1, also presynaptic Arp2 and Arp3 are crucial for the formation of type Ib synaptic boutons. Interestingly, presynaptic Abp1 functions in NMJ formation were fully dependent on the Arp2/3 complex, as revealed by suppression of Abp1-induced synaptic bouton formation and branching of axon terminals upon presynaptic Arp2 RNAi. These data reveal that Abp1 and Arp2/3 complex-mediated actin cytoskeletal dynamics drive both synaptic bouton formation and NMJ branching. Our data furthermore shed light on an intense bidirectional functional crosstalk between pre- and postsynapses during the development of synaptic contacts.

Lack of evidence of the interaction of the Aß peptide with the Wnt signaling cascade in Drosophila models of Alzheimer¿s disease

BackgroundAlzheimer's disease (AD) is the leading form of dementia worldwide. The Aβ-peptide is believed to be the major pathogenic compound of the disease. Since several years it is hypothesized that Aβ impacts the Wnt signaling cascade and therefore activation of this signaling pathway is proposed to rescue the neurotoxic effect of Aβ.FindingsExpression of the human Aβ42 in the Drosophila nervous system leads to a drastically shortened life span. We found that the action of Aβ42 specifically in the glutamatergic motoneurons is responsible for the reduced survival. However, we find that the morphology of the glutamatergic larval neuromuscular junctions, which are widely used as the model for mammalian central nervous system synapses, is not affected by Aβ42 expression. We furthermore demonstrate that genetic activation of the Wnt signal transduction pathway in the nervous system is not able to rescue the shortened life span or a rough eye phenotype in Drosophila.ConclusionsOur data confirm that the life span is a useful readout of Aβ42 induced neurotoxicity in Drosophila; the neuromuscular junction seems however not to be an appropriate model to study AD in flies. Additionally, our results challenge the hypothesis that Wnt signaling might be implicated in Aβ42 toxicity and might serve as a drug target against AD.

Part-based motor neuron recognition in the Drosophila ventral nerve cord

We exploit the morphological stereotypy and relative simplicity of the Drosophila nervous system to model the diverse neuronal morphologies of individual motor neurons and understand underlying principles of synaptic connectivity in a motor circuit. In our analysis, we use images depicting single neurons labeled with green fluorescent protein (GFP) and serially imaged with laser scanning confocal microscopy. We model morphology with a novel formulation of Conditional Random Fields, a hierarchical latent-state CRF, to capture the highly varying compartment-based structure of the neurons (soma–axon–dendrites). In the training phase, we follow two approaches: (i) hierarchical learning, where compartment labels are given, and (ii) latent-state learning, where compartment labels are not given in the samples. We demonstrate the accuracy of our approach using wild-type motor neurons in the larval ventral nerve cord. However, our method can also be used for the identification of motor neuron mutations, as well as the automated annotation of the motor circuitry in wild type and mutant animals. Our method is directly applicable to the recognition of compartment-defined structures.

The pre‐synaptic Munc13‐1 binds alcohol and modulates alcohol self‐administration in Drosophila

Munc13-1 is a pre-synaptic active-zone protein essential for neurotransmitter release and involved in pre-synaptic plasticity in brain. Ethanol, butanol, and octanol quenched the intrinsic fluorescence of the C1 domain of Munc13-1 with EC₅₀ s of 52 mM, 26 mM, and 0.7 mM, respectively. Photoactive azialcohols photolabeled Munc13-1 C1 exclusively at Glu-582, which was identified by mass spectrometry. Mutation of Glu-582 to alanine, leucine, and histidine reduced the alcohol binding two- to five-fold. Circular dichroism studies suggested that binding of alcohol increased the stability of the wild-type Munc13-1 compared with the mutants. If Munc13-1 plays some role in the neural effects of alcohol in vivo, changes in the activity of this protein should produce differences in the behavioral responses to ethanol. We tested this prediction with a loss-of-function mutation in the conserved Dunc-13 in Drosophila melanogaster. The Dunc-13(P84200) /+ heterozygotes have 50% wild-type levels of Dunc-13 mRNA and display a very robust increase in ethanol self-administration. This phenotype is reversed by the expression of the rat Munc13-1 protein within the Drosophila nervous system. The present studies indicate that Munc13-1 C1 has binding site(s) for alcohols and Munc13-1 activity is sufficient to restore normal self-administration to Drosophila mutants deficient in Dunc-13 activity. The pre-synaptic Mun13-1 protein is a critical regulator of synaptic vesicle fusion and may be involved in processes that lead to ethanol abuse and addiction. We studied its interaction with alcohol and identified Glu-582 as a critical residue for ethanol binding. Munc13-1 can functionally complement the Dunc13 haploinsufficient ethanol self-administration phenotype in Drosophila melanogaster, indicating that this protein participates in alcohol-induced behavioral plasticity.