Drosophila enabled promotes synapse morphogenesis and regulates active zone form and function.

BACKGROUND: Recent studies of synapse form and function highlight the importance of the actin cytoskeleton in regulating multiple aspects of morphogenesis, neurotransmission, and neural plasticity. The conserved actin-associated protein Enabled (Ena) is known to regulate development of the Drosophila larval neuromuscular junction through a postsynaptic mechanism. However, the functions and regulation of Ena within the presynaptic terminal has not been determined. METHODS: Here, we use a conditional genetic approach to address a presynaptic role for Ena on presynaptic morphology and ultrastructure, and also examine the pathway in which Ena functions through epistasis experiments. RESULTS: We find that Ena is required to promote the morphogenesis of presynaptic boutons and branches, in contrast to its inhibitory role in muscle. Moreover, while postsynaptic Ena is regulated by microRNA-mediated mechanisms, presynaptic Ena relays the output of the highly conserved receptor protein tyrosine phosphatase Dlar and associated proteins including the heparan sulfate proteoglycan Syndecan, and the non-receptor Abelson tyrosine kinase to regulate addition of presynaptic varicosities. Interestingly, Ena also influences active zones, where it restricts active zone size, regulates the recruitment of synaptic vesicles, and controls the amplitude and frequency of spontaneous glutamate release. CONCLUSION: We thus show that Ena, under control of the Dlar pathway, is required for presynaptic terminal morphogenesis and bouton addition and that Ena has active zone and neurotransmission phenotypes. Notably, in contrast to Dlar, Ena appears to integrate multiple pathways that regulate synapse form and function.

Target-dependent retrograde signaling mediates synaptic plasticity at the Drosophila neuromuscular junction.

Neurons that innervate multiple targets often establish synapses with target-specific strengths, and local forms of synaptic plasticity. We have examined the molecular-genetic mechanisms that allow a single Drosophila motoneuron, the ventral Common Exciter (vCE), to establish connections with target-specific properties at its various synaptic partners. By driving transgenes in a subset of vCE's targets, we found that individual target cells are able to independently control the properties of vCE's innervating branch and synapses. This is achieved by means of a trans-synaptic growth factor secreted by the target cell. At the larval neuromuscular junction, postsynaptic glutamate receptor activity stimulates the release of the BMP4/5/6 homolog Glass bottom boat (Gbb). As larvae mature and motoneuron terminals grow, Gbb activates the R-Smad transcriptional regulator phosphorylated Mad (pMad) to facilitate presynaptic development. We found that manipulations affecting glutamate receptors or Gbb within subsets of target muscles led to local effects either specific to the manipulated muscle or by a limited gradient within the presynaptic branches. While presynaptic development depends on pMad transcriptional activity within the motoneuron nucleus, we find that the Gbb growth factor may also act locally within presynaptic terminals. Local Gbb signaling and presynaptic pMad accumulation within boutons may therefore participate in a "synaptic tagging" mechanism, to influence synaptic growth and plasticity in Drosophila.

Activity-Dependent Synaptic Refinement: New Insights from Drosophila.

During development, neurons establish inappropriate connections as they seek out their synaptic partners, resulting in supernumerary synapses that must be pruned away. The removal of miswired synapses usually involves electrical activity, often through a Hebbian spike-timing mechanism. A novel form of activity-dependent refinement is used by Drosophila that may be non-Hebbian, and is critical for generating the precise connectivity observed in that system. In Drosophila, motoneurons use both glutamate and the biogenic amine octopamine for neurotransmission, and the muscle fibers receive multiple synaptic inputs. Motoneuron growth cones respond in a time-regulated fashion to multiple chemotropic signals arising from their postsynaptic partners. Central to this mechanism is a very low frequency (<0.03 Hz) oscillation of presynaptic cytoplasmic calcium, that regulates and coordinates the action of multiple downstream effectors involved in the withdrawal from off-target contacts. Low frequency calcium oscillations are widely observed in developing neural circuits in mammals, and have been shown to be critical for normal connectivity in a variety of neural systems. In Drosophila these mechanisms allow the growth cone to sample widely among possible synaptic partners, evaluate opponent chemotropic signals, and withdraw from off-target contacts. It is possible that the underlying molecular mechanisms are conserved widely among invertebrates and vertebrates.

In Vivo Calcium Signaling during Synaptic Refinement at the Drosophila Neuromuscular Junction.

Neural activity plays a key role in pruning aberrant synapses in various neural systems, including the mammalian cortex, where low-frequency (0.01 Hz) calcium oscillations refine topographic maps. However, the activity-dependent molecular mechanisms remain incompletely understood. Activity-dependent pruning also occurs at embryonic Drosophila neuromuscular junctions (NMJs), where low-frequency Ca2+ oscillations are required for synaptic refinement and the response to the muscle-derived chemorepellant Sema2a. We examined embryonic growth cone filopodia in vivo to directly observe their exploration and to analyze the episodic Ca2+ oscillations involved in refinement. Motoneuron filopodia repeatedly contacted off-target muscle fibers over several hours during late embryogenesis, with episodic Ca2+ signals present in both motile filopodia as well as in later-stabilized synaptic boutons. The Ca2+ transients matured over several hours into regular low-frequency (0.03 Hz) oscillations. In vivo imaging of intact embryos of both sexes revealed that the formation of ectopic filopodia is increased in Sema2a heterozygotes. We provide genetic evidence suggesting a complex presynaptic Ca2+-dependent signaling network underlying refinement that involves the phosphatases calcineurin and protein phosphatase-1, as well the serine/threonine kinases CaMKII and PKA. Significantly, this network influenced the neuron's response to the muscle's Sema2a chemorepellant, critical for the removal of off-target contacts.SIGNIFICANCE STATEMENT To address the question of how synaptic connectivity is established during development, we examined the behavior of growth cone filopodia during the exploration of both correct and off-target muscle fibers in Drosophila embryos. We demonstrate that filopodia repeatedly contact off-target muscles over several hours, until they ultimately retract. We show that intracellular signals are observed in motile and stabilized "ectopic" contacts. Several genetic experiments provide insight in the molecular pathway underlying network refinement, which includes oscillatory calcium signals via voltage-gated calcium channels as a key component. Calcium orchestrates the activity of several kinases and phosphatases, which interact in a coordinated fashion to regulate chemorepulsion exerted by the muscle.

Cyclic nucleotide signaling is required during synaptic refinement at the Drosophila neuromuscular junction.

The removal of miswired synapses is a fundamental prerequisite for normal circuit development, leading to clinical problems when aberrant. However, the underlying activity-dependent molecular mechanisms involved in synaptic pruning remain incompletely resolved. Here the dynamic properties of intracellular calcium oscillations and a role for cAMP signaling during synaptic refinement in intact Drosophila embryos were examined using optogenetic tools. We provide In vivo evidence at the single gene level that the calcium-dependent adenylyl cyclase rutabaga, the phosphodiesterase dunce, the kinase PKA, and Protein Phosphatase 1 (PP1) all operate within a functional signaling pathway to modulate Sema2a-dependent chemorepulsion. It was found that presynaptic cAMP levels were required to be dynamically maintained at an optimal level to suppress connectivity defects. It was also proposed that PP1 may serve as a molecular link between cAMP signaling and CaMKII in the pathway underlying refinement. The results introduced an in vivo model where presynaptic cAMP levels, downstream of electrical activity and calcium influx, act via PKA and PP1 to modulate the neuron's response to chemorepulsion involved in the withdrawal of off-target synaptic contacts. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 39-60, 2017.

Mutations in KATNB1 cause complex cerebral malformations by disrupting asymmetrically dividing neural progenitors.

Exome sequencing analysis of over 2,000 children with complex malformations of cortical development identified five independent (four homozygous and one compound heterozygous) deleterious mutations in KATNB1, encoding the regulatory subunit of the microtubule-severing enzyme Katanin. Mitotic spindle formation is defective in patient-derived fibroblasts, a consequence of disrupted interactions of mutant KATNB1 with KATNA1, the catalytic subunit of Katanin, and other microtubule-associated proteins. Loss of KATNB1 orthologs in zebrafish (katnb1) and flies (kat80) results in microcephaly, recapitulating the human phenotype. In the developing Drosophila optic lobe, kat80 loss specifically affects the asymmetrically dividing neuroblasts, which display supernumerary centrosomes and spindle abnormalities during mitosis, leading to cell cycle progression delays and reduced cell numbers. Furthermore, kat80 depletion results in dendritic arborization defects in sensory and motor neurons, affecting neural architecture. Taken together, we provide insight into the mechanisms by which KATNB1 mutations cause human cerebral cortical malformations, demonstrating its fundamental role during brain development.

A systematic nomenclature for the insect brain.

Despite the importance of the insect nervous system for functional and developmental neuroscience, descriptions of insect brains have suffered from a lack of uniform nomenclature. Ambiguous definitions of brain regions and fiber bundles have contributed to the variation of names used to describe the same structure. The lack of clearly determined neuropil boundaries has made it difficult to document precise locations of neuronal projections for connectomics study. To address such issues, a consortium of neurobiologists studying arthropod brains, the Insect Brain Name Working Group, has established the present hierarchical nomenclature system, using the brain of Drosophila melanogaster as the reference framework, while taking the brains of other taxa into careful consideration for maximum consistency and expandability. The following summarizes the consortium's nomenclature system and highlights examples of existing ambiguities and remedies for them. This nomenclature is intended to serve as a standard of reference for the study of the brain of Drosophila and other insects.

Retrograde BMP signaling at the synapse: a permissive signal for synapse maturation and activity-dependent plasticity.

At the Drosophila neuromuscular junction (NMJ), the loss of retrograde, trans-synaptic BMP signaling causes motoneuron terminals to have fewer synaptic boutons, whereas increased neuronal activity results in a larger synapse with more boutons. Here, we show that an early and transient BMP signal is necessary and sufficient for NMJ growth as well as for activity-dependent synaptic plasticity. This early critical period was revealed by the temporally controlled suppression of Mad, the SMAD1 transcriptional regulator. Similar results were found by genetic rescue tests involving the BMP4/5/6 ligand Glass bottom boat (Gbb) in muscle, and alternatively the type II BMP receptor Wishful Thinking (Wit) in the motoneuron. These observations support a model where the muscle signals back to the innervating motoneuron's nucleus to activate presynaptic programs necessary for synaptic growth and activity-dependent plasticity. Molecular genetic gain- and loss-of-function studies show that genes involved in NMJ growth and plasticity, including the adenylyl cyclase Rutabaga, the Ig-CAM Fasciclin II, the transcription factor AP-1 (Fos/Jun), and the adhesion protein Neurexin, all depend critically on the canonical BMP pathway for their effects. By contrast, elevated expression of Lar, a receptor protein tyrosine phosphatase found to be necessary for activity-dependent plasticity, rescued the phenotypes associated with the loss of Mad signaling. We also find that synaptic structure and function develop using genetically separable, BMP-dependent mechanisms. Although synaptic growth depended on Lar and the early, transient BMP signal, the maturation of neurotransmitter release was independent of Lar and required later, ongoing BMP signaling.

Genetic systems for functional cell ablation in Drosophila.

The selective removal of cells by ablation is a powerful tool in the study of eukaryotic developmental biology, providing much information about the origin, fate, or function of these cells in the developing organism. In Drosophila, three main methods have been used to ablate cells: chemical, genetic, and laser ablation. Each method has its own applicability with regard to developmental stage and the cells to be ablated, and its own limitations. This article describes genetic systems for functional cell ablation in Drosophila. Genetic ablation consists of delivering a toxin or death-inducing gene under the control of a cell-specific enhancer, or by means of the GAL4 system. Because of the wide range of existing enhancers, toxins and death genes can be targeted to virtually any cell of choice, allowing for cell-type-specificity. Genetic ablation is less expensive and less labor-intensive than laser ablation. It allows one to analyze the effects of eliminating every cell of a given type within an embryo, and also allows the examination of populations rather than individuals.

TUNEL-antibody double-labeling method for Drosophila embryos.

The terminal deoxynucleotide transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method for monitoring targeted cell ablation is based on the in situ labeling of DNA fragmentation sites in nuclei of intact fixed cells. Unlike other methods of detecting dying cells, the use of fixed material allows antigen expression to be monitored at the same time that apoptosis is confirmed in the targeted cells. Double-labeling of Drosophila embryos using the TUNEL reaction and fluorescently tagged antibodies can be adapted to the selected antigen. For some antigens, it is preferable that the TUNEL reaction be performed first, whereas for others, the TUNEL reaction should follow antigen detection. This may be because some antigens may not survive the 37°C incubation or the conditions of the reaction. Similarly, increased fixation times yield better results for some antigens, but not for others. This protocol describes a TUNEL reaction adapted for use on Drosophila embryos in conjunction with fluorescently labeled antibodies.

Setup for functional cell ablation with lasers: coupling of a laser to a microscope.

The selective removal of cells by ablation is a powerful tool in the study of eukaryotic developmental biology, providing much information about their origin, fate, or function in the developing organism. In Drosophila, three main methods have been used to ablate cells: chemical, genetic, and laser ablation. Each method has its own applicability with regard to developmental stage and the cells to be ablated, and its own limitations. The primary advantage of laser-based ablation is the flexibility provided by the method: The operations can be performed in any cell pattern and at any time in development. Laser-based techniques permit manipulation of structures within cells, even to the molecular level. They can also be used for gene activation. However, laser ablation can be expensive, labor-intensive, and time-consuming. Although live cells can be difficult to image in Drosophila embryos, the use of vital fluorescent imaging methods has made laser-mediated cell manipulation methods more appealing; the methods are relatively straightforward. This article provides the information necessary for setting up and using a laser microscope for lasesr ablation studies.

Embryonic cell ablation in Drosophila using lasers.

Cell ablation is a powerful tool in the study of eukaryotic developmental biology. The selective removal of cells by ablation may provide much information about their origin, fate, or function in the developing organism. Laser-based techniques have an advantage over genetic or chemical ablation methods in that the operations can be performed in essentially any cell pattern and at any time in development. This protocol describes the methods needed to target and ablate specific cells of interest in Drosophila embryos with lasers.

Antibody staining of the central nervous system in adult Drosophila.

The Drosophila nervous system provides a valuable model for studying various aspects of brain development and function. The postembryonic Drosophila brain is especially useful, because specific neuron types derive from specific progenitors at specific times. Elucidating the means by which diverse neuron types derive from a limited number of progenitors can contribute significantly to our understanding of the genetic and molecular mechanisms involved in developmental neurobiology. Antibody-labeling techniques are particularly useful for examining the Drosophila brain. These methods generally use primary antibodies specific to a protein or a structure of interest and a fluorescently labeled or enzyme-coupled secondary antibody to detect the primary antibodies. Immunofluorescence methods allow for simultaneous probing for multiple antigens using different fluorophores, as well as high-resolution confocal examination of deep structures. This protocol describes general procedures for antibody labeling of neural tissue from Drosophila, as well as visualization techniques for fluorescent and enzyme-linked probes.

X-gal staining of the central nervous system in adult Drosophila.

The Drosophila nervous system provides a valuable model for studying various aspects of brain development and function. The postembryonic Drosophila brain is especially useful, because specific neuron types derive from specific progenitors at specific times. Elucidating the means by which diverse neuron types derive from a limited number of progenitors can contribute significantly to our understanding of the genetic and molecular mechanisms involved in developmental neurobiology. ß-Galactosidase, the product of the E. coli lacZ gene, has been used extensively as a reporter in Drosophila research. Staining for ß-galactosidase activity can be performed using the substrate X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside), which produces a blue precipitate visible by light microscopy. This detection method is highly sensitive and has the advantage that the results can be observed without the need for specialized microscopy equipment. This protocol describes general procedures for X-gal labeling of neural tissue from Drosophila.

Hydroxyurea ablation of mushroom bodies in Drosophila.

Chemical ablation is an effective tool for studying nervous system development and function in Drosophila. Hydroxyurea (HU) inhibits ribonucleotide reductase, blocking DNA synthesis, and killing dividing cells. The specificity of HU ablation is thus dependent on developmental events. In this respect, HU is useful in determining temporal patterns of neuroblast proliferation and the origins of neuronal elements in flies and other insects. In Drosophila, an especially fortuitous time window occurs at the end of embryonic development. For the first 8-12 h after larval hatching, only five neuroblasts are proliferating in each brain hemisphere. Four of these are found in the dorsal protocerebrum and give rise to the intrinsic elements (Kenyon cells [KCs] and glia) of the mushroom bodies (MBs). The remaining single neuroblast has an anterolateral position in the brain and is the progenitor of local interneurons (LocI) in the antennal lobe (AL) and a subset of lateral relay interneurons (RIl) in the inner antennocerebral tract (iACT). Treating newly hatched larvae with HU results in adult flies with KCs and AL interneurons of embryonic origin only. This protocol describes methods for collecting newly hatched Drosophila larvae and treating them with HU.

Experimental methods for examining synaptic plasticity in Drosophila.

The Drosophila neuromuscular junction (NMJ) ranks as one of the preeminent model systems for studying synaptic development, function, and plasticity. In this article, we review the experimental genetic methods that include the use of mutated or reengineered ion channels to manipulate the synaptic connections made by motor neurons onto larval body-wall muscles. We also provide a consideration of environmental and rearing conditions that phenocopy some of the genetic manipulations.

Monitoring membrane excitability in Drosophila expressing modified shaker constructs.

The Drosophila neuromuscular junction (NMJ) ranks as one of the preeminent model systems for studying synaptic development, function, and plasticity. This protocol describes the use of the two-electrode voltage clamp (TEVC) to examine potassium (K(+)) currents mediated by voltage-gated ion channels, and gives several genetic and pharmacological methods that are used to study the currents. Drosophila larval muscle fibers possess three major K(+) currents. One of these, a fast voltage-activating and inactivating I(A) current, is mediated by the Shaker channel. The Shaker channel is characterized by its sensitivity to the drug 4-aminopyridine (4-AP). Two useful transgenic tools for altering membrane excitability have been developed by making specific modifications of the Shaker channel; their use is described here.

Dissection of adult Drosophila brains.

The Drosophila nervous system provides a valuable model for studying various aspects of brain development and function. The postembryonic Drosophila brain is especially useful, because specific neuron types derive from specific progenitors at particular times. Elucidating the means by which diverse neuron types derive from a limited number of progenitors can contribute significantly to our understanding of the genetic and molecular mechanisms involved in developmental neurobiology. This protocol describes general procedures for dissecting the brain and ventral nerve cord (VNC) of adult Drosophila. The dissected tissues are suitable for further analysis, e.g., by any number of labeling techniques.

Cracking the combinatorial semaphorin code.

In this issue of Neuron, Wu et al. describe a combinatorial code of repulsive Sema-2a and attractive Sema-2b signaling that mediates mechanosensory axonal guidance, fasciculation, and synaptic target selection within the CNS of Drosophila. Their work exemplifies how a detailed, multilevel molecular-genetic analysis (from molecules to behavior) provides fundamental insights into neural circuit development.