The matricellular protein Drosophila Cellular Communication Network Factor is required for synaptic transmission and female fertility.

Within the extracellular matrix, matricellular proteins are dynamically expressed nonstructural proteins that interact with cell surface receptors, growth factors, and proteases, as well as with structural matrix proteins. The cellular communication network factors family of matricellular proteins serve regulatory roles to regulate cell function and are defined by their conserved multimodular organization. Here, we characterize the expression and neuronal requirement for the Drosophila cellular communication network factor family member. Drosophila cellular communication network factor is expressed in the nervous system throughout development including in subsets of monoamine-expressing neurons. Drosophila cellular communication network factor-expressing abdominal ganglion neurons innervate the ovaries and uterus and the loss of Drosophila cellular communication network factor results in reduced female fertility. In addition, Drosophila cellular communication network factor accumulates at the synaptic cleft and is required for neurotransmission at the larval neuromuscular junction. Analyzing the function of the single Drosophila cellular communication network factor family member will enhance our potential to understand how the microenvironment impacts neurotransmitter release in distinct cellular contexts and in response to activity.

Intact Drosophila central nervous system cellular quantitation reveals sexual dimorphism.

Establishing with precision the quantity and identity of the cell types of the brain is a prerequisite for a detailed compendium of gene and protein expression in the central nervous system (CNS). Currently, however, strict quantitation of cell numbers has been achieved only for the nervous system of Caenorhabditis elegans. Here, we describe the development of a synergistic pipeline of molecular genetic, imaging, and computational technologies designed to allow high-throughput, precise quantitation with cellular resolution of reporters of gene expression in intact whole tissues with complex cellular constitutions such as the brain. We have deployed the approach to determine with exactitude the number of functional neurons and glia in the entire intact larval Drosophila CNS, revealing fewer neurons and more glial cells than previously predicted. We also discover an unexpected divergence between the sexes at this juvenile developmental stage, with the female CNS having significantly more neurons than that of males. Topological analysis of our data establishes that this sexual dimorphism extends to deeper features of CNS organisation. We additionally extended our analysis to quantitate the expression of voltage-gated potassium channel family genes throughout the CNS and uncover substantial differences in abundance. Our methodology enables robust and accurate quantification of the number and positioning of cells within intact organs, facilitating sophisticated analysis of cellular identity, diversity, and gene expression characteristics.

A conditional GABAergic synaptic vesicle marker for Drosophila.

BACKGROUND: Throughout the animal kingdom, GABA is the principal inhibitory neurotransmitter of the nervous system. It is essential for maintaining the homeostatic balance between excitation and inhibition required for the brain to operate normally. Identification of GABAergic neurons and their GABA release sites are thus essential for understanding how the brain regulates the excitability of neurons and the activity of neural circuits responsible for numerous aspects of brain function including information processing, locomotion, learning, memory, and synaptic plasticity, among others. NEW METHOD: Since the structure and features of GABA synapses are critical to understanding their function within specific neural circuits of interest, here we developed and characterized a conditional marker of GABAergic synaptic vesicles for Drosophila, 9XV5-vGAT. RESULTS: 9XV5-vGAT is validated for conditionality of expression, specificity for localization to synaptic vesicles, specificity for expression in GABAergic neurons, and functionality. Its utility for GABAergic neurotransmitter phenotyping and identification of GABA release sites was verified for ellipsoid body neurons of the central complex. In combination with previously reported conditional SV markers for acetylcholine and glutamate, 9XV5-vGAT was used to demonstrate fast neurotransmitter phenotyping of subesophageal ganglion neurons. COMPARISON WITH EXISTING METHODS: This method is an alternative to single cell transcriptomics for neurotransmitter phenotyping and can be applied to any neurons of interest represented by a binary transcription system driver. CONCLUSION: A conditional GABAergic synaptic vesicle marker has been developed and validated for GABA neurotransmitter phenotyping and subcellular localization of GABAergic synaptic vesicles.

A conditional glutamatergic synaptic vesicle marker for Drosophila.

Glutamate is a principal neurotransmitter used extensively by the nervous systems of all vertebrate and invertebrate animals. It is primarily an excitatory neurotransmitter that has been implicated in nervous system development, as well as a myriad of brain functions from the simple transmission of information between neurons to more complex aspects of nervous system function including synaptic plasticity, learning, and memory. Identification of glutamatergic neurons and their sites of glutamate release are thus essential for understanding the mechanisms of neural circuit function and how information is processed to generate behavior. Here, we describe and characterize smFLAG-vGlut, a conditional marker of glutamatergic synaptic vesicles for the Drosophila model system. smFLAG-vGlut is validated for functionality, conditional expression, and specificity for glutamatergic neurons and synaptic vesicles. The utility of smFLAG-vGlut is demonstrated by glutamatergic neurotransmitter phenotyping of 26 different central complex neuron types of which nine were established to be glutamatergic. This illumination of glutamate neurotransmitter usage will enhance the modeling of central complex neural circuitry and thereby our understanding of information processing by this region of the fly brain. The use of smFLAG for glutamatergic neurotransmitter phenotyping and identification of glutamate release sites can be extended to any Drosophila neuron(s) represented by a binary transcription system driver.

Miniature neurotransmission is required to maintain Drosophila synaptic structures during ageing.

The decline of neuronal synapses is an established feature of ageing accompanied by the diminishment of neuronal function, and in the motor system at least, a reduction of behavioural capacity. Here, we have investigated Drosophila motor neuron synaptic terminals during ageing. We observed cumulative fragmentation of presynaptic structures accompanied by diminishment of both evoked and miniature neurotransmission occurring in tandem with reduced motor ability. Through discrete manipulation of each neurotransmission modality, we find that miniature but not evoked neurotransmission is required to maintain presynaptic architecture and that increasing miniature events can both preserve synaptic structures and prolong motor ability during ageing. Our results establish that miniature neurotransmission, formerly viewed as an epiphenomenon, is necessary for the long-term stability of synaptic connections.

Characterization of Drosophila octopamine receptor neuronal expression using MiMIC-converted Gal4 lines.

Octopamine, the invertebrate analog of norepinephrine, is known to modulate a large variety of behaviors in Drosophila including feeding initiation, locomotion, aggression, and courtship, among many others. Significantly less is known about the identity of the neurons that receive octopamine input and how they mediate octopamine-regulated behaviors. Here, we characterize adult neuronal expression of MiMIC-converted Trojan-Gal4 lines for each of the five Drosophila octopamine receptors. Broad neuronal expression was observed for all five octopamine receptors, yet distinct differences among them were also apparent. Use of immunostaining for the octopamine neurotransmitter synthesis enzyme Tdc2, along with a novel genome-edited conditional Tdc2-LexA driver, revealed all five octopamine receptors express in Tdc2/octopamine neurons to varying degrees. This suggests autoreception may be an important circuit mechanism by which octopamine modulates behavior.

Octopamine neuron dependent aggression requires dVGLUT from dual-transmitting neurons.

Neuromodulators such as monoamines are often expressed in neurons that also release at least one fast-acting neurotransmitter. The release of a combination of transmitters provides both "classical" and "modulatory" signals that could produce diverse and/or complementary effects in associated circuits. Here, we establish that the majority of Drosophila octopamine (OA) neurons are also glutamatergic and identify the individual contributions of each neurotransmitter on sex-specific behaviors. Males without OA display low levels of aggression and high levels of inter-male courtship. Males deficient for dVGLUT solely in OA-glutamate neurons (OGNs) also exhibit a reduction in aggression, but without a concurrent increase in inter-male courtship. Within OGNs, a portion of VMAT and dVGLUT puncta differ in localization suggesting spatial differences in OA signaling. Our findings establish a previously undetermined role for dVGLUT in OA neurons and suggests that glutamate uncouples aggression from OA-dependent courtship-related behavior. These results indicate that dual neurotransmission can increase the efficacy of individual neurotransmitters while maintaining unique functions within a multi-functional social behavior neuronal network.

Demonstration of a Simple Epitope Tag Multimerization Strategy for Enhancing the Sensitivity of Protein Detection Using Drosophila vAChT.

The expression and distribution of a protein can provide critical information about its function in a cell. For some neuronal proteins this information may include neurotransmitter (NT) usage and sites of NT release. However, visualizing the expression of a protein within a given neuron is often challenging because most neurons are intricately intermingled with numerous other neurons, making individual neuronal expression difficult to discern, especially since many neuronal genes are expressed at low levels. To overcome these difficulties for the Drosophila vesicular acetylcholine transporter (vAChT), attempts were made to generate conditional Drosophila vAChT alleles containing two tandem copies of epitope tags. In the course of these attempts, a strategy for multimerizing DNA repeats using the Gibson cloning reaction was serendipitously discovered. Attempts at optimization routinely yielded six or seven copies of MYC and OLLAS epitope tag coding sequences, but occasionally as many as 10 copies, thus potentially enhancing the sensitivity of protein detection up to an order of magnitude. As proof-of-principle of the method, conditionally expressible genome-edited 7XMYC-vAChT and 6XOLLAS-vAChT were developed and characterized for conditionality, synaptic vesicle specificity, and neurotransmitter specific-expression. The utility of these conditional vAChT variants was demonstrated for cholinergic neurotransmitter phenotyping and defining the polarity of cholinergic neurons, important information for understanding the functional role of neurons of interest in neural circuits and behavior. The repeat multimerization method is effective for DNA repeats of at least 56 bp and should be generally applicable to any species.

Conditional Synaptic Vesicle Markers for Drosophila.

The release of neurotransmitters from synaptic vesicles (SVs) at pre-synaptic release sites is the principle means by which information transfer between neurons occurs. Knowledge of the location of SVs within a neuron can thus provide valuable clues about the location of neurotransmitter release within a neuron and the downstream neurons to which a given neuron is connected, important information for understanding how neural circuits generate behavior. Here the development and characterization of four conditional tagged SV markers for Drosophila melanogaster is presented. This characterization includes evaluation of conditionality, specificity for SV localization, and sensitivity of detection in diverse neuron subtypes. These four SV markers are genome-edited variants of the synaptic vesicle-specific protein Rab3. They depend on either the B2 or FLP recombinases for conditionality, and incorporate GFP or mCherry fluorescent proteins, or FLAG or HA epitope tags, for detection.

t-GRASP, a targeted GRASP for assessing neuronal connectivity.

BACKGROUND: Understanding how behaviors are generated by neural circuits requires knowledge of the synaptic connections between the composite neurons. Methods for mapping synaptic connections, such as electron microscopy and paired recordings, are labor intensive and alternative methods are thus desirable. NEW METHOD: Development of a targeted GFP Reconstitution Across Synaptic Partners(GRASP) method, t-GRASP, for assessing neural connectivity is described. RESULTS: Numerous different pre-synaptic and post-synaptic/dendritic proteins were tested for enhancing the specificity of GRASP signal to synaptic regions. Pairing of both targeted pre- and post-t-GRASP constructs resulted in strong preferential GRASP signal in synaptic regions in Drosophila larval sensory neurons, larval neuromuscular junctions, and adult photoreceptor neurons with minimal false-positive signal. COMPARISON WITH EXISTING METHODS: Activity-independent t-GRASP exhibits an enhancement of GRASP signal specificity for synaptic contact sites as compared to existing Drosophila GRASP methods. Fly strains were developed for expression of both pre- and post-t-GRASP with each of the three Drosophila binary transcription systems, thus enabling GRASP assays to be performed between any two driver pairs of any transcription system in either direction, an option not available for existing Drosophila GRASP methods. CONCLUSIONS: t-GRASP is a novel targeted GRASP method for assessing synaptic connectivity between Drosophila neurons. Its flexibility of use with all three Drosophila binary transcription systems significantly expands the potential use of GRASP in Drosophila.

Hexameric GFP and mCherry reporters for the Drosophila GAL4, Q, and LexA transcription systems.

The ability to distinguish cells and tissues of interest is critical for understanding their biological importance. In genetic model organisms, a prominent approach for discerning particular cells or tissues from others is the use of cell or tissue-specific enhancers to drive fluorescent reporters. This approach, however, is often limited by the brightness of the fluorescent reporter. To augment the ability to visualize cells or tissues of interest in Drosophila melanogaster, homo-hexameric GFP and mCherry reporters were developed for the GAL4, Q, and LexA transcription systems and functionally validated in vivo. The GFP and mCherry homo-hexameric fusion proteins exhibited significantly enhanced fluorescence as compared to monomeric fluorescent reporters and could be visualized by direct fluorescence throughout the cytoplasm of neurons, including the fine processes of axons and dendrites. These high-sensitivity fluorescent reporters of cell morphology can be utilized for a variety of purposes, especially facilitating fluorescence-based genetic screens for cell morphology phenotypes. These results suggest that the strategy of fusing monomeric fluorescent proteins in tandem to enhance brightness should be generalizable to other fluorescent proteins and other genetic model organisms.

Expansion of the gateway multisite recombination cloning toolkit.

Precise manipulation of transgene expression in genetic model organisms has led to advances in understanding fundamental mechanisms of development, physiology, and genetic disease. Transgene construction is, however, a precondition of transgene expression, and often limits the rate of experimental progress. Here we report an expansion of the modular Gateway MultiSite recombination-cloning platform for high efficiency transgene assembly. The expansion includes two additional destination vectors and entry clones for the LexA binary transcription system, among others. These new tools enhance the expression levels possible with Gateway MultiSite generated transgenes and make possible the generation of LexA drivers and reporters with Gateway MultiSite cloning. In vivo data from transgenic Drosophila functionally validating each novel component are presented and include neuronal LexA drivers, LexAop2 red and green fluorescent synaptic vesicle reporters, TDC2 and TRH LexA, GAL4, and QF drivers, and LexAop2, UAS, and QUAS channelrhodopsin2 T159C reporters.

A Gateway MultiSite recombination cloning toolkit.

The generation of DNA constructs is often a rate-limiting step in conducting biological experiments. Recombination cloning of single DNA fragments using the Gateway system provided an advance over traditional restriction enzyme cloning due to increases in efficiency and reliability. Here we introduce a series of entry clones and a destination vector for use in two, three, and four fragment Gateway MultiSite recombination cloning whose advantages include increased flexibility and versatility. In contrast to Gateway single-fragment cloning approaches where variations are typically incorporated into model system-specific destination vectors, our Gateway MultiSite cloning strategy incorporates variations in easily generated entry clones that are model system-independent. In particular, we present entry clones containing insertions of GAL4, QF, UAS, QUAS, eGFP, and mCherry, among others, and demonstrate their in vivo functionality in Drosophila by using them to generate expression clones including GAL4 and QF drivers for various trp ion channel family members, UAS and QUAS excitatory and inhibitory light-gated ion channels, and QUAS red and green fluorescent synaptic vesicle markers. We thus establish a starter toolkit of modular Gateway MultiSite entry clones potentially adaptable to any model system. An inventory of entry clones and destination vectors for Gateway MultiSite cloning has also been established (www.gatewaymultisite.org).

An efficient method for recombineering GAL4 and QF drivers.

Neural circuit mapping and manipulation are facilitated by independent control of gene expression in pre- and post-synaptic neurons. The GAL4/UAS and Q binary transcription systems have the potential to provide this capability. Of particular use in neural circuit mapping would be GAL4 and QF drivers specific for neurotransmitters and neurotransmitter receptors. Recently available Drosophila genomic BAC libraries make recombineering large genes including those specific for neurotransmitters and neurotransmitter receptors feasible. Here the functionality of cassettes that allow efficient recombineering of GAL4 and QF drivers based on kanamycin selection is demonstrated in Drosophila. The cassettes should, however, be generalizable for recombineering in other species.

Red blood cell transfusion in a patient with anti-AnWj: a case report.

Anti-AnWj (Anton) has been associated with clinically significant hemolytic transfusion reactions. More than 99 percent of studied populations have RBCs that express the antigen. Reported here is a patient with anti-AnWj who was transfused with antigen-positive RBCs without adverse reaction.

Drosophila huntingtin-interacting protein 14 is a presynaptic protein required for photoreceptor synaptic transmission and expression of the palmitoylated proteins synaptosome-associated protein 25 and cysteine string protein.

Palmitoylation affects the trafficking, stability, aggregation, and/or functional activity of a substantial number of neuronal proteins. We identified mutations in dHIP14, the Drosophila homolog of the human palmitoyl transferase, Huntingtin-interacting protein 14 (HIP14). HIP14 was previously reported to localize primarily to Golgi and to palmitoylate the neuronal proteins synaptosome-associated protein 25 (SNAP-25), PSD-95 (postsynaptic density-95), GAD65, Synaptotagmin, and Huntingtin in mammalian neurons. We find dHIP14 to be an essential maternal effect gene required for photoreceptor synaptic transmission and for proper in vivo expression of the palmitoylated presynaptic proteins SNAP-25 and cysteine string protein. In non-neuronal cells in the fly, dHIP14 protein is found in Golgi. However, in fly neurons, we find dHIP14 primarily in presynaptic terminals, something we also observe with HIP14. In mammalian neurons, we also find a significant fraction of HIP14 colocalizing with a synaptic vesicle marker. Based on localization of the palmitoyl transferase HIP14 within the presynaptic nerve terminal, we propose palmitoylation as a possible mechanism that may be operating to rapidly regulate synaptic efficacy.

Roles for Drp1, a dynamin-related protein, and milton, a kinesin-associated protein, in mitochondrial segregation, unfurling and elongation during Drosophila spermatogenesis.

Mitochondria undergo dramatic rearrangement during Drosophila spermatogenesis. In wild type testes, the many small mitochondria present in pre-meiotic spermatocytes later aggregate, fuse, and interwrap in post-meiotic haploid spermatids to form the spherical Nebenkern, whose two giant mitochondrial compartments later unfurl and elongate beside the growing flagellar axoneme. Drp1 encodes a dynamin-related protein whose homologs in many organisms mediate mitochondrial fission and whose Drosophila homolog is known to govern mitochondrial morphology in neurons. The milton gene encodes an adaptor protein that links mitochondria with kinesin and that is required for mitochondrial transport in Drosophila neurons. To determine the roles of Drp1 and Milton in spermatogenesis, we used the FLP-FRT mitotic recombination system to generate spermatocytes homozygous for mutations in either gene in an otherwise heterozygous background. We found that absence of Drp1 leads to abnormal clustering of mitochondria in mature primary spermatocytes and aberrant unfurling of the mitochondrial derivatives in early Drp1 spermatids undergoing axonemal elongation. In milton spermatocytes, mitochondria are distributed normally; however, after meiosis, the Nebenkern is not strongly anchored to the nucleus, and the mitochondrial derivatives do not elongate properly. Our work defines specific functions for Drp1 and Milton in the anchoring, unfurling, and elongation of mitochondria during sperm formation.

Axonal transport of mitochondria requires milton to recruit kinesin heavy chain and is light chain independent.

Mitochondria are distributed within cells to match local energy demands. We report that the microtubule-dependent transport of mitochondria depends on the ability of milton to act as an adaptor protein that can recruit the heavy chain of conventional kinesin-1 (kinesin heavy chain [KHC]) to mitochondria. Biochemical and genetic evidence demonstrate that kinesin recruitment and mitochondrial transport are independent of kinesin light chain (KLC); KLC antagonizes milton's association with KHC and is absent from milton-KHC complexes, and mitochondria are present in klc (-/-) photoreceptor axons. The recruitment of KHC to mitochondria is, in part, determined by the NH(2) terminus-splicing variant of milton. A direct interaction occurs between milton and miro, which is a mitochondrial Rho-like GTPase, and this interaction can influence the recruitment of milton to mitochondria. Thus, milton and miro are likely to form an essential protein complex that links KHC to mitochondria for light chain-independent, anterograde transport of mitochondria.