Publications

49 publications

Filopodial Dynamics and Growth Cone Stabilization in Drosophila Visual Circuit Development

Ozel, M.N., Langen, M., Hassan, B.A. and Hiesinger, P.R.

eLife Sciences Publications | 2015-10-29

Appeared In: eLife 2015;4:e10721

Full text [PDF]

Filopodial dynamics are thought to control growth cone guidance, but the types and roles of growth cone dynamics underlying neural circuit assembly in a living brain are largely unknown. To address this issue, we have developed long-term, continuous, fast and high-resolution imaging of growth cone dynamics from axon growth to synapse formation in cultured Drosophila brains. Using R7 photoreceptor neurons as a model we show that >90% of the growth cone filopodia exhibit fast, stochastic dynamics that persist despite ongoing stepwise layer formation. Correspondingly, R7 growth cones stabilize early and change their final position by passive dislocation. N-Cadherin controls both fast filopodial dynamics and growth cone stabilization. Surprisingly, loss of N-Cadherin causes no primary targeting defects, but destabilizes R7 growth cones to jump between correct and incorrect layers. Hence, growth cone dynamics can influence wiring specificity without a direct role in target recognition and implement simple rules during circuit assembly.

Subject: D. melanogaster; brain development; filopodial dynamics; growth cone; live Imaging; neuroscience; synapse formation

Beyond Molecular Codes: Simple Rules to Wire Complex Brain

Hassan, B.A. and Hiesinger, P.R.

Cell Press | 2015-10-08

Appeared In: Cell, 163(2):285-291

http://dx.doi.org/10.1016/j.cell.2015.09 ...

Molecular codes, like postal zip codes, are generally considered a robust way to ensure the specificity of neuronal target selection. However, a code capable of unambiguously generating complex neural circuits is difficult to conceive. Here, we re-examine the notion of molecular codes in the light of developmental algorithms. We explore how molecules and mechanisms that have been considered part of a code may alternatively implement simple pattern formation rules sufficient to ensure wiring specificity in neural circuits. This analysis delineates a pattern-based framework for circuit construction that may contribute to our understanding of brain wiring.

The Developmental Rules of Neural Superposition in Drosophila.

Langen, M.*, Agi, E.*, Altschuler, D., Wu, L.*, Altschuler, S.*, and Hiesinger, P.R.*

Cell Press | 2015-06-25

Appeared In: Cell 162(1): 120-33

http://dx.doi.org/10.1016/j.cell.2015.05 ...

Complicated neuronal circuits can be genetically encoded, but the underlying developmental algorithms remain largely unknown. Here, we describe a developmental algorithm for the specification of synaptic partner cells through axonal sorting in the Drosophila visual map. Our approach combines intravital imaging of growth cone dynamics in developing brains of intact pupae and data-driven computational modeling. These analyses suggest that three simple rules are sufficient to generate the seemingly complex neural superposition wiring of the fly visual map without an elaborate molecular matchmaking code. Our computational model explains robust and precise wiring in a crowded brain region despite extensive growth cone overlaps and provides a framework for matching molecular mechanisms with the rules they execute. Finally, ordered geometric axon terminal arrangements that are not required for neural superposition are a side product of the developmental algorithm, thus elucidating neural circuit connectivity that remained unexplained based on adult structure and function alone. PAPERCLIP.

The evolution and development of neural superposition.

Agi, E., Langen, M., Altschuler, S., Wu, L., Zimmermann, T., and Hiesinger, P.R.

Informa Healthcare | 2014-07-08

Appeared In: J. Neurogenet. 28(3-4): 216-32

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Visual systems have a rich history as model systems for the discovery and understanding of basic principles underlying neuronal connectivity. The compound eyes of insects consist of up to thousands of small unit eyes that are connected by photoreceptor axons to set up a visual map in the brain. The photoreceptor axon terminals thereby represent neighboring points seen in the environment in neighboring synaptic units in the brain. Neural superposition is a special case of such a wiring principle, where photoreceptors from different unit eyes that receive the same input converge upon the same synaptic units in the brain. This wiring principle is remarkable, because each photoreceptor in a single unit eye receives different input and each individual axon, among thousands others in the brain, must be sorted together with those few axons that have the same input. Key aspects of neural superposition have been described as early as 1907. Since then neuroscientists, evolutionary and developmental biologists have been fascinated by how such a complicated wiring principle could evolve, how it is genetically encoded, and how it is developmentally realized. In this review article, we will discuss current ideas about the evolutionary origin and developmental program of neural superposition. Our goal is to identify in what way the special case of neural superposition can help us answer more general questions about the evolution and development of genetically "hard-wired" synaptic connectivity in the brain.

Subject: Drosophila, Visual System, synapse, brain wiring

Regulation of branching dynamics by axon-intrinsic asymmetries in Tyrosine Kinase Receptor signaling.

Zschatzsch, M., Oliva, C., Langen, M., De Geest, N., Ozel, M.N., Williamson , W.R., Lemon, W., Soldano, A., Munck, S., Hiesinger P.R., Sanchez-Soriano, N., Hassan, B.

eLife Sciences Publications, Ltd. | 2014-04-22

Appeared In: Elife 3: e01699

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Axonal branching allows a neuron to connect to several targets, increasing neuronal circuit complexity. While axonal branching is well described, the mechanisms that control it remain largely unknown. We find that in the Drosophila CNS branches develop through a process of excessive growth followed by pruning. In vivo high-resolution live imaging of developing brains as well as loss and gain of function experiments show that activation of Epidermal Growth Factor Receptor (EGFR) is necessary for branch dynamics and the final branching pattern. Live imaging also reveals that intrinsic asymmetry in EGFR localization regulates the balance between dynamic and static filopodia. Elimination of signaling asymmetry by either loss or gain of EGFR function results in reduced dynamics leading to excessive branch formation. In summary, we propose that the dynamic process of axon branch development is mediated by differential local distribution of signaling receptors. DOI: http://dx.doi.org/10.7554/eLife.01699.001.

Subject: axonal branching; brain development; signaling

Calcium/Calmodulin regulates SNARE assembly and spontaneous neurotransmitter release via v-ATPase subunit V0a1

Wang, D., Epstein, D., Khalaf, O., Srinivasan, S., Williamson, W.R., Fayyazuddin, A., Quiocho, F.A., and Hiesinger, P.R.

Rockefeller University Press | 2014-04-14

Appeared In: J. Cell. Biol. 205(1):21-31

Full text [PDF]Suppl. mat. [PDF]

Most chemical neurotransmission occurs through Ca2+-dependent evoked or spontaneous vesicle exocytosis. In both cases, Ca2+ sensing is thought to occur shortly before exocytosis. In this paper, we provide evidence that the Ca2+ dependence of spontaneous vesicle release may partly result from an earlier requirement of Ca2+ for the assembly of soluble N-ethylmaleimide–sensitive fusion attachment protein receptor (SNARE) complexes. We show that the neuronal vacuolar-type H+-adenosine triphosphatase V0 subunit a1 (V100) can regulate the formation of SNARE complexes in a Ca2+–Calmodulin (CaM)-dependent manner. Ca2+–CaM regulation of V100 is not required for vesicle acidification. Specific disruption of the Ca2+-dependent regulation of V100 by CaM led to a >90% loss of spontaneous release but only had a mild effect on evoked release at Drosophila melanogaster embryo neuromuscular junctions. Our data suggest that Ca2+–CaM regulation of V100 may control SNARE complex assembly for a subset of synaptic vesicles that sustain spontaneous release.

Charcot-Marie-Tooth 2B mutations in rab7 cause dosage-dependent neurodegeneration due to partial loss of function.

Cherry, S., Jin, E.J., Ozel, M.N., Lu, Z., Agi, E., Wang, D., Meinertzhagen, I.A., Chan, C.-C., and Hiesinger, P.R.

eLife Sciences Publications, Ltd. | 2013-12-10

Appeared In: Elife 2: e01064

Full text [PDF]

The small GTPase Rab7 is a key regulator of endosomal maturation in eukaryotic cells. Mutations in rab7 are thought to cause the dominant neuropathy Charcot-Marie-Tooth 2B (CMT2B) by a gain-of-function mechanism. Here we show that loss of rab7, but not overexpression of rab7 CMT2B mutants, causes adult-onset neurodegeneration in a Drosophila model. All CMT2B mutant proteins retain 10-50% function based on quantitative imaging, electrophysiology, and rescue experiments in sensory and motor neurons in vivo. Consequently, expression of CMT2B mutants at levels between 0.5 and 10-fold their endogenous levels fully rescues the neuropathy-like phenotypes of the rab7 mutant. Live imaging reveals that CMT2B proteins are inefficiently recruited to endosomes, but do not impair endosomal maturation. These findings are not consistent with a gain-of-function mechanism. Instead, they indicate a dosage-dependent sensitivity of neurons to rab7-dependent degradation. Our results suggest a therapeutic approach opposite to the currently proposed reduction of mutant protein function. DOI: http://dx.doi.org/10.7554/eLife.01064.001.

Subject: endosome; genetics; neuropathy; synapse

The vesicular ATPase: a missing link between acidification and exocytosis.

Wang, D. and Hiesinger, P.R.

Rockefeller University Press | 2013-10-28

Appeared In: J. Cell Biol. 203(2): 171-3

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The vesicular adenosine triphosphatase (ATPase) acidifies intracellular compartments, including synaptic vesicles and secretory granules. A controversy about a second function of this ATPase in exocytosis has been fuelled by questions about multiple putative roles of acidification in the exocytic process. Now, Poëa-Guyon et al. (2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201303104) present new evidence that the vesicular ATPase performs separate acidification and exocytosis roles and propose a mechanism for how these two functions are causally linked.

Recombineering Homologous Recombination Constructs in Drosophila.

Carrerira-Rosario, A., Scoggin, S., Shalaby, N.A., Williams, N.D., Hiesinger, P.R. and Buszczak, M.

MYJoVE Corporation | 2013-07-13

Appeared In: J Vis Exp. (77): 50346

Video linkFull text [PDF]

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Subject: Genetics, Issue 77, Bioengineering, Molecular Biology, Biomedical Engineering, Physiology, Drosophila melanogaster, genetics (animal and plant), Recombineering, Drosophila, Homologous Recombination, Knock-out, recombination, genetic engineering, gene targeting, gene, genes, DNA, PCR, Primers, sequencing, animal model

The synaptic maintenance problem: membrane recycling, Ca2+ homeostasis and late onset degeneration.

Bezprozvanny, I., and Hiesinger, P.R

BioMed Central | 2013-07-08

Appeared In: Mol Neurodegener 8: 23

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Most neurons are born with the potential to live for the entire lifespan of the organism. In addition, neurons are highly polarized cells with often long axons, extensively branched dendritic trees and many synaptic contacts. Longevity together with morphological complexity results in a formidable challenge to maintain synapses healthy and functional. This challenge is often evoked to explain adult-onset degeneration in numerous neurodegenerative disorders that result from otherwise divergent causes. However, comparably little is known about the basic cell biological mechanisms that keep normal synapses alive and functional in the first place. How the basic maintenance mechanisms are related to slow adult-onset degeneration in different diseasesis largely unclear. In this review we focus on two basic and interconnected cell biological mechanisms that are required for synaptic maintenance: endomembrane recycling and calcium (Ca(2+)) homeostasis. We propose that subtle defects in these homeostatic processes can lead to late onset synaptic degeneration. Moreover, the same basic mechanisms are hijacked, impaired or overstimulated in numerous neurodegenerative disorders. Understanding the pathogenesis of these disorders requires an understanding of both the initial cause of the disease and the on-going changes in basic maintenance mechanisms. Here we discuss the mechanisms that keep synapses functional over long periods of time with the emphasis on their role in slow adult-onset neurodegeneration.

Subject: Neurodegeneration, Endosome, Autophagy, Alzheimer’s disease, Calcium, Presenilin, Amyloid, Huntington’s disease, Hereditary motor and sensory neuropathy, Lysosomal storage disorder, Ataxia, Calcineurin, Excitotoxicity

Membrane trafficking in Neuronal Maintenance and Degeneration.

Wang, D., Chan, C.C., Cherry, S. and Hiesinger, P.R.

Springer Science+Business Media | 2012-11-08

Appeared In: Cell. Mol. Life Sci., 70(16):2919-34

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Defects in membrane trafficking and degradation are hallmarks of most, and maybe all, neurodegenerative disorders. Such defects typically result in the accumulation of undegraded proteins due to aberrant endosomal sorting, lysosomal degradation, or autophagy. The genetic or environmental cause of a specific disease may directly affect these membrane trafficking processes. Alternatively, changes in intracellular sorting and degradation can occur as cellular responses of degenerating neurons to unrelated primary defects such as insoluble protein aggregates or other neurotoxic insults. Importantly, altered membrane trafficking may contribute to the pathogenesis or indeed protect the neuron. The observation of dramatic changes to membrane trafficking thus comes with the challenging need to distinguish pathological from protective alterations. Here, we will review our current knowledge about the protective and destructive roles of membrane trafficking in neuronal maintenance and degeneration. In particular, we will first focus on the question of what type of membrane trafficking keeps healthy neurons alive in the first place. Next, we will discuss what alterations of membrane trafficking are known to occur in Alzheimer’s disease and other tauopathies, Parkinson’s disease, polyQ diseases, peripheral neuropathies, and lysosomal storage disorders. Combining the maintenance and degeneration viewpoints may yield insight into how to distinguish when membrane trafficking functions protectively or contributes to degeneration.

Subject: Autophagy, Endosome, Lysosome, Huntington, Alzheimer, Parkinson

Similarities of Drosophila rab GTPases based on expression profiling: completion and analysis of the rab-Gal4 kit.

Jin, E.J., Chan, C.C., Agi, E., Cherry, S., Hanacik, E., Buszczak, M., and Hiesinger, P.R.

Public Library of Science | 2012-07-23

Appeared In: PLoS ONE 7(7): e40912

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We recently generated rab-Gal4 lines for 25 of 29 predicted Drosophila rab GTPases. These lines provide tools for the expression of reporters, mutant rab variants or other genes, under control of the regulatory elements of individual rab loci. Here, we report the generation and characterization of the remaining four rab-Gal4 lines. Based on the completed 'rab-Gal4 kit' we performed a comparative analysis of the cellular and subcellular expression of all rab GTPases. This analysis includes the cellular expression patterns in characterized neuronal and non-neuronal cells and tissues, the subcellular localization of wild type, constitutively active and dominant negative rab GTPases and colocalization with known intracellular compartment markers. Our comparative analysis identifies all Rab GTPases that are expressed in the same cells and localize to the same intracellular compartments. Remarkably, similarities based on these criteria are typically not predicted by primary sequence homology. Hence, our findings provide an alternative basis to assess potential roles and redundancies based on expression in developing and adult cell types, compartment identity and subcellular localization.

Lysosomal calcium homeostasis defects, not proton pump defects, cause endo-lysosomal dysfunction in PSEN-deficient cells.

Coen, K. Flannagan, R.S., Carraro-Lacroix, L.R., Wang, D., Vermeire, W., Michiels, C. Munck, S., Sugita, S., Hiesinger, P.R. Grinstein, S., Annaert, W.

Rockefeller University Press | 2012-07-09

Appeared In: J. Cell Biol., 198(1):23-35

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Presenilin (PSEN) deficiency is accompanied by accumulation of endosomes and autophagosomes, likely caused by impaired endo-lysosomal fusion. Recently, Lee et al. (2010. Cell. doi: http://dx.doi.org/10.1016/j.cell.2010.05.008) attributed this phenomenon to PSEN1 enabling the transport of mature V0a1 subunits of the vacuolar ATPase (V-ATPase) to lysosomes. In their view, PSEN1 mediates the N-glycosylation of V0a1 in the endoplasmic reticulum (ER); consequently, PSEN deficiency prevents V0a1 glycosylation, compromising the delivery of unglycosylated V0a1 to lysosomes, ultimately impairing V-ATPase function and lysosomal acidification. We show here that N-glycosylation is not a prerequisite for proper targeting and function of this V-ATPase subunit both in vitro and in vivo in Drosophila melanogaster. We conclude that endo-lysosomal dysfunction in PSEN−/− cells is not a consequence of failed N-glycosylation of V0a1, or compromised lysosomal acidification. Instead, lysosomal calcium storage/release is significantly altered in PSEN−/− cells and neurons, thus providing an alternative hypothesis that accounts for the impaired lysosomal fusion capacity and accumulation of endomembranes that accompanies PSEN deficiency.

Subcellular Resolution Imaging in Neural Circuits. Bookchapter in: The Making and Un-Making of Neuronal Circuits

Williamson, W.R., Chan, C.C., and Hiesinger, P.R.

Humana Press | 2012-05-23

Appeared In: The Making and Un-Making of Neuronal Circuits in Drosophila. Volume 69 of the series Neuromethods. pp 61-89

http://dx.doi.org/10.1007/978-1-61779-83 ...

Drosophila combines advanced genetics with a brain of ideal size for high-resolution imaging in toto. However, imaging of intracellular compartments pushes the limits of light microscopy in every system, and at the subcellular level the small size of fly neurons presents a challenge. In this chapter, we review recent imaging advances that, often for the first time, allow the visualization of intracellular biology of neurons in the context of their neuronal circuits. We discuss the different preparations that keep neural circuit architectures intact for live and fixed imaging. Finally, we review advances in light microscopy and imaging probes in combination with these preparations and provide a guide to which high-resolution microscopy techniques are applicable to the different Drosophila preparations. We focus on the imaging of intracellular membrane trafficking dynamics. However, since any imaging of intracellular trafficking constitutes an example of imaging at subcellular resolution, many approaches discussed here will be useful for the study of neuronal cell biology in Drosophila in general.

Subject: Fluorescent microscopy, High-resolution imaging, Brain dissection, Immunohis-tochemistry, Live imaging

Autophagy, Neuron-Specific Degradation and Neurodegeneration.

Wang, D., and Hiesinger, P.R.

Landes Bioscience | 2012-04-01

Appeared In: Autophagy, 8(4):711-3

Full text [PDF]

Degradation of membrane compartments, organelles and other debris through macroautophagy (hereafter referred to as autophagy) is thought to occur in most, maybe all, cells. We recently reported the discovery of a neuron-specific endomembrane degradation mechanism that depends on the vesicle SNARE neuronal Synaptobrevin (n-Syb) and the vesicle ATPase component V100 (the V0a1 subunit). Loss of n-Syb causes degeneration of adult photoreceptor neurons in Drosophila, reminiscent of adult-onset degeneration in neurons with defective autophagy. Here we explore the potential importance of this newly discovered neuron-specific degradation mechanism in comparison with ubiquitous autophagy machinery for adult-onset neurodegeneration.

Subject: Drosophila, endocytic trafficking, neurodegeneration, SNARE, v-ATPase

Combining recombineering and ends-out homologous recombination to systematically characterize Drosophila gene families: Rab GTPases as a case study.

Chan, C.C., Scoggin, S., Hiesinger, P.R.* and Buszczak, M.*

Taylor & Francis | 2012-03-01

Appeared In: Commun Integr Biol 5(2): 179-83

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Evaluating how an individual gene contributes to a particular biological process benefits greatly from a comprehensive understanding of all members of its gene family. Such knowledge is ideally obtained using multicellular model organisms, which provide rapid and decisive platforms for determining gene function. We recently established a novel transgenesis platform in Drosophila to systematically knock out all members of the Rab small GTPase family of membrane regulators. This platform combines BAC transgenesis/recombineering with ends-out homologous recombinations and Gateway(TM) technologies and provides a new rapid and scalable method that eases the manipulation of endogenous loci. This method not only allows for the generation of molecularly defined lesions, but also the precise replacement or tagging of genes in their endogenous loci. Using this method, we found that up to half of all Rab GTPases exhibit enriched expression at synapses in the nervous system. Here we provide critical details about the underlying recombineering and transgenesis method, new cassettes for tagging endogenous loci and information on important parameters that will allow Drosophila researchers to target members of other gene families.

Subject: Drosophila; Rab GTPase; gene targeting; homologous recombination; recombineering

The synaptic vesicle SNARE neuronal Synaptobrevin promotes endolysosomal degradation and prevents neurodegeneration in Drosophila.

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Haberman, A.S., Williamson, W.R., Epstein, D., Wang, D., Rina, S., Meinertzhagen, I.A. and Hiesinger, P.R.

Rockefeller University Press | 2012-01-23

Appeared In: J. Cell. Biol. 196(2): 261-276

Full text [PDF]Suppl. mat. [PDF]

Soluble NSF attachment protein receptors (SNAREs) are the core proteins in membrane fusion. The neuron-specific synaptic v-SNARE n-syb (neuronal Synaptobrevin) plays a key role during synaptic vesicle exocytosis. In this paper, we report that loss of n-syb caused slow neurodegeneration independent of its role in neurotransmitter release in adult Drosophila melanogaster photoreceptor neurons. In addition to synaptic vesicles, n-Syb localized to endosomal vesicles. Loss of n-syb lead to endosomal accumulations, transmembrane protein degradation defects, and a secondary increase in autophagy. Our evidence suggests a primary defect of impaired delivery of vesicles that contain degradation proteins, including the acidification-activated Cathepsin proteases and the neuron-specific proton pump and V0 adenosine triphosphatase component V100. Overexpressing V100 partially rescued n-syb–dependent degeneration through an acidification-independent endosomal sorting mechanism. Collectively, these findings reveal a role for n-Syb in a neuron-specific sort-and-degrade mechanism that protects neurons from degeneration. Our findings further shed light on which intraneuronal compartments exhibit increased or decreased neurotoxicity.

Systematic discovery of Rab GTPases with synaptic functions in Drosophila.

Chan, C.C., Scoggin, S., Wang, D., Cherry, S., Dembo, T., Greenberg, B., Jin, E.J., Kuey, C., Lopez, A., Mehta, S.Q., Perkins, T.J., Brankatschk, M., Rothenfluh, A., Buszczak, M.*, and Hiesinger, P.R.*

Cell Press | 2011-10-13

Appeared In: Curr. Biol. 21(20): 1704-15

Full text [PDF]Suppl. mat. [PDF]

Neurons require highly specialized intracellular membrane trafficking, especially at synapses. Rab GTPases are considered master regulators of membrane trafficking in all cells, and only very few Rabs have known neuron-specific functions. Here, we present the first systematic characterization of neuronal expression, subcellular localization, and function of Rab GTPases in an organism with a brain.

A Drosophila genetic screen yields allelic series of core microRNA biogenesis factors and reveals post-developmental roles for microRNAs.

Smibert, P., Bejarano, F., Wang, D., Garaulet, D.L., Yang, J.-S., Martin, R., Bortolamiol-Becet, D., Robine, N., Hiesinger, P.R. and Lai, E.C..

Cold Spring Harbor Laboratory Press | 2011-09-23

Appeared In: RNA 17(11): 1997-2010

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Canonical animal microRNAs (miRNAs) are ∼22-nt regulatory RNAs generated by stepwise cleavage of primary hairpin transcripts by the Drosha and Dicer RNase III enzymes. We performed a genetic screen using an miRNA-repressed reporter in the Drosophila eye and recovered the first reported alleles of fly drosha, an allelic series of its dsRBD partner pasha, and novel alleles of dicer-1. Analysis of drosha mutants provided direct confirmation that mirtrons are independent of this nuclease, as inferred earlier from pasha knockouts. We further used these mutants to demonstrate in vivo cross-regulation of Drosha and Pasha in the intact animal, confirming remarkable conservation of a homeostatic mechanism that aligns their respective levels. Although the loss of core miRNA pathway components is universally lethal in animals, we unexpectedly recovered hypomorphic alleles that gave adult escapers with overtly normal development. However, the mutant photoreceptor neurons exhibited reduced synaptic transmission, without accompanying defects in neuronal development or maintenance. These findings indicate that synaptic function is especially sensitive to optimal miRNA pathway function. These allelic series of miRNA pathway mutants should find broad usage in studies of miRNA biogenesis and biology in the Drosophila system.

Subject: microRNA, Drosha, Pasha, Microprocessor, synapse, Dicer

Intracellular trafficking in Drosophila visual system development: a basis for pattern formation through simple mechanisms.

Chan, C.C., Epstein, D., and Hiesinger, P.R.

Wiley Subscription Services, Inc. | 2011-06-28

Appeared In: Dev Neurobiol 71(12): 1227-45

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Intracellular trafficking underlies cellular functions ranging from membrane remodeling to receptor activation. During multicellular organ development, these basic cell biological functions are required as both passive machinery and active signaling regulators. Exocytosis, endocytosis, and recycling of several key signaling receptors have long been known to actively regulate morphogenesis and pattern formation during Drosophila eye development. Hence, intracellular membrane trafficking not only sets the cell biological stage for receptor-mediated signaling but also actively controls signaling through spatiotemporally regulated receptor localization. In contrast to eye development, the role of intracellular trafficking for the establishment of the eye-to-brain connectivity map has only recently received more attention. It is still poorly understood how guidance receptors are spatiotemporally regulated to serve as meaningful synapse formation signals. Yet, the Drosophila visual system provides some of the most striking examples for the regulatory role of intracellular trafficking during multicellular organ development. In this review we will first highlight the experimental and conceptual advances that motivate the study of intracellular trafficking during Drosophila visual system development. We will then illuminate the development of the eye, the eye-to-brain connectivity map and the optic lobe from the perspective of cell biological dynamics. Finally, we provide a conceptual framework that seeks to explain how the interplay of simple genetically encoded intracellular trafficking events governs the seemingly complex cellular behaviors, which in turn determine the developmental product.

Subject: receptor sorting; degradation; signaling; neurogenetics; optic lobe

Guidance receptor degradation is required for neuronal connectivity in the Drosophila nervous system.

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Williamson, W.R., Yang, T., Terman, J.R., and Hiesinger, P.R.

Public Library of Science | 2010-12-07

Appeared In: PLoS Biol. 8(12): e1000553

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Axon pathfinding and synapse formation rely on precise spatiotemporal localization of guidance receptors. However, little is known about the neuron-specific intracellular trafficking mechanisms that underlie the sorting and activity of these receptors. Here we show that loss of the neuron-specific v-ATPase subunit a1 leads to progressive endosomal guidance receptor accumulations after neuronal differentiation. In the embryo and in adult photoreceptors, these accumulations occur after axon pathfinding and synapse formation is complete. In contrast, receptor missorting occurs sufficiently early in neurons of the adult central nervous system to cause connectivity defects. An increase of guidance receptors, but not of membrane proteins without signaling function, causes specific gain-of-function phenotypes. A point mutant that promotes sorting but prevents degradation reveals spatiotemporally specific guidance receptor turnover and accelerates developmental defects in photoreceptors and embryonic motor neurons. Our findings indicate that a neuron-specific endolysosomal degradation mechanism is part of the cell biological machinery that regulates guidance receptor turnover and signaling.

On the role of v-ATPase V0a1-dependent degradation in Alzheimer disease.

Williamson, W.R. and Hiesinger, P.R.

Taylor & Francis | 2010-11-01

Appeared In: Commun Integr Biol 3(6): 604-7

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Defective autophagy and lysosomal degradation are hallmarks of numerous neurodegenerative disorders. Vesicular ATPases are intracellular proton pumps that acidify autophagosomes and lysosomes. V0a1 is a key component of the v-ATPase that is only required in neurons in Drosophila melanogaster. We have recently shown that loss of V0a1 in Drosophila photoreceptor neurons leads to slow, adult-onset degeneration.1 Concurrently, Lee et al.2 reported that V0a1 fails to localize to lysosomal compartments in cells from Presenilin 1 knock-out cells. Together these two reports suggest that a neuronal V0a1-dependent degradation mechanism may be causally linked to Alzheimer pathology. Indeed, we now show that loss of V0a1 makes Drosophila neurons more susceptible to insult with human Alzheimer-related neurotoxic Aβ and tau proteins. Furthermore, we discuss the potential significance of the discovery of the neuron-specific degradation mechanism in Drosophila for intracellular degradation defects in Alzheimer Disease.

Subject: Neurodegeneration, Autophagy, degradation, acidification, vesicular ATPase, Alzheimer

A dual function of V0-ATPase a1 provides an endolysosomal degradation mechanism in Drosophila photoreceptors.

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Williamson, W.R., Wang, D., Haberman, A.S. and Hiesinger, P.R.

Rockefeller University Press | 2010-05-31

Appeared In: J. Cell. Biol., 189: 885-99

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The vesicular adenosine triphosphatase (v-ATPase) is a proton pump that acidifies intracellular compartments. In addition, mutations in components of the membrane-bound v-ATPase V0 sector cause acidification-independent defects in yeast, worm, fly, zebrafish, and mouse. In this study, we present a dual function for the neuron-specific V0 subunit a1 orthologue v100 in Drosophila melanogaster. A v100 mutant that selectively disrupts proton translocation rescues a previously characterized synaptic vesicle fusion defect and vesicle fusion with early endosomes. Correspondingly, V100 selectively interacts with syntaxins on the respective target membranes, and neither synaptic vesicles nor early endosomes require v100 for their acidification. In contrast, V100 is required for acidification once endosomes mature into degradative compartments. As a consequence of the complete loss of this neuronal degradation mechanism, photoreceptors undergo slow neurodegeneration, whereas selective rescue of the acidification-independent function accelerates cell death by increasing accumulations in degradation-incompetent compartments. We propose that V100 exerts a temporally integrated dual function that increases neuronal degradative capacity.

Preparation of developing and adult Drosophila brains and retinae for live imaging.

Williamson, W.R. and Hiesinger, P.R.

MYJoVE Corporation | 2010-03-15

Appeared In: J Vis Exp (37):

Full text [PDF]Video link

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Subject: Neuroscience, Issue 37, dissection technique, eye disc, brain culture, photoreceptor, confocal microscopy

Visual System Development: Invertebrates

Hiesinger, P.R. and Meinertzhagen, I.A.

Elesevier | 2009

Appeared In: Squire LR (ed.) Encyclopedia of Neuroscience, volume 10, pp. 313-322. Oxford: Academic Press, Elsevier

http://dx.doi.org/10.1007/978-1-4899-677 ...

The visual systems of most invertebrate groups arise following the outgrowth of axons from photoreceptors which develop from the ectodermal epithelium independently of the nerve centers which they innervate; peripherally derived visual interneurons are exceptional. This is in contrast to vertebrates where the retina forms from an embryological outgrowth of the forebrain, with several classes of interneurons. Most studies on invertebrates are descriptive and preferentially treat eye development. Few studies are analytical and fewer treat the visual centers. Yet the topographic ordering, the small numbers of their identifiable neurons, and the many other model characteristics of invertebrate neuropils especially recommend these centers for studies in neural development and regeneration, notably the compound eyes of arthropods and the highly developed single-lens eyes of certain molluscs. Modern studies have tended to canonize selected species (especially the flies Drosophila and Musca, and the water-flea Daphnia) to the unwarranted neglect of other equally deserving groups—cephalopods, pectinid scallops, spiders, and salps to name but a few. A comprehensive review by Meinertzhagen and Macagno is forthcoming.

Synaptic patterning by morphogen signaling.

Williamson, W.R. and Hiesinger, P.R.

American Association for the Advancement of Science | 2008-05-06

Appeared In: Sci Signal 1(18): pe20

http://dx.doi.org/10.1126/stke.118pe20

Gradients of secreted small morphogenic molecules control cell proliferation and patterning throughout animal development. Recent years have seen the discovery of surprising roles for morphogens in later developmental processes, including axon pathfinding and synaptogenesis. The latest addition is a role for the TGF-beta superfamily morphogen Activin in synaptic patterning of the Drosophila visual system. In contrast to classical instructive and long-range morphogen gradients, Activin acts as a permissive and local motility restriction signal around several hundred individual photoreceptor axon terminals. Activin must therefore act in concert with other instructively attracting and repelling signals as part of a larger genetic program for brain wiring.

NAD synthase NMNAT acts as a chaperone to protect against neurodegeneration.

Zhai, R.G., Zhang, F., Hiesinger, P.R., Cao, Y., Haeuter, C.M., and Bellen, H.J.

Nature Publishing Group | 2008-03-16

Appeared In: Nature 452(7189): 887-91

http://dx.doi.org/10.1038/nature06721

Neurodegeneration can be triggered by genetic or environmental factors. Although the precise cause is often unknown, many neurodegenerative diseases share common features such as protein aggregation and age dependence. Recent studies in Drosophila have uncovered protective effects of NAD synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) against activity-induced neurodegeneration and injury-induced axonal degeneration. Here we show that NMNAT overexpression can also protect against spinocerebellar ataxia 1 (SCA1)-induced neurodegeneration, suggesting a general neuroprotective function of NMNAT. It protects against neurodegeneration partly through a proteasome-mediated pathway in a manner similar to heat-shock protein 70 (Hsp70). NMNAT displays chaperone function both in biochemical assays and cultured cells, and it shares significant structural similarity with known chaperones. Furthermore, it is upregulated in the brain upon overexpression of poly-glutamine expanded protein and recruited with the chaperone Hsp70 into protein aggregates. Our results implicate NMNAT as a stress-response protein that acts as a chaperone for neuronal maintenance and protection. Our studies provide an entry point for understanding how normal neurons maintain activity, and offer clues for the common mechanisms underlying different neurodegenerative conditions.

Optic Lobe Development, in Brain Development of Drosophia

Fischbach, K.F. and Hiesinger, P.R.

Springer | 2008

Appeared In: Ed. G. Technau, Landes Biosciences.

http://dx.doi.org/10.1007/978-0-387-7826 ...

The optic lobes comprise approximately half of the fly’s brain. In four major synaptic ganglia, or neuropils, the visual input from the compound eyes is received and processed for higher order visual functions like motion detection and color vision. A common characteristic of vertebrate and invertebrate visual systems is the point-to-point mapping of the visual world to synaptic layers in the brain, referred to as visuotopy. Vision requires the parallel extraction of numerous parameters in a visuotopic manner. Consequently, the optic neuropils are arranged in columns and perpendicularly oriented synaptic layers that allow for the selective establishment of synapses between columnar neurons. How this exquisite synaptic specificity is established during approximately 100 hours of brain development is still poorly understood. However, the optic lobe contains one of the best characterized brain structures in any organism—both anatomically and developmentally. Moreover, numerous molecules and their function illuminate some of the basic mechanisms involved in brain wiring. The emerging picture is that the development of the visual system of Drosophila is (epi-)genetically hard-wired; it supplies the emerging fly with vision without requiring neuronal activity for fine tuning of neuronal connectivity. Elucidating the genetic and cellular principles by which gene activity directs the assembly of the optic lobe is therefore a fascinating task and the focus of this chapter.

V-ATPase V0 sector subunit a1 in neurons is a target of calmodulin.

Zhang, W., Wang, D., Volk, E., Bellen, H.J., Hiesinger, P.R.*, Quiocho, F.A.* , * co-corresponding authors

American Society for Biochemistry and Molecular Biology | 2007-10-12

Appeared In: J. Biol. Chem. 283(1): 294-300

Full text [PDF]

The V(0) complex forms the proteolipid pore of a vesicular ATPase that acidifies vesicles. In addition, an independent function in membrane fusion has been suggested in vacuolar fusion in yeast and synaptic vesicle exocytosis in fly neurons. Evidence for a direct role in secretion has also recently been presented in mouse and worm. The molecular mechanisms of how the V(0) components might act or are regulated are largely unknown. Here we report the identification and characterization of a calmodulin-binding site in the large cytosolic N-terminal region of the Drosophila protein V100, the neuron-specific V(0) subunit a1. V100 forms a tight complex with calmodulin in a Ca(2+)-dependent manner. Mutations in the calmodulin-binding site in Drosophila lead to a loss of calmodulin recruitment to synapses. Neuronal expression of a calmodulin-binding deficient V100 uncovers an incomplete rescue at low levels and cellular toxicity at high levels. Our results suggest a vesicular ATPase V(0)-dependent function of calmodulin at synapses.

Thirty-one flavors of Drosophila rab proteins.

Zhang, J., Schulze, K.L., Hiesinger, P.R., Suyama, K., Wang, S., Fish, M., Acar, M., Hoskins, R.A., Bellen, H.J., and Scott, M.P.

Genetics Society Of America | 2007-03-04

Appeared In: Genetics 176(2): 1307-22

Full text [PDF]Suppl. mat. [PDF]

Rab proteins are small GTPases that play important roles in transport of vesicle cargo and recruitment, association of motor and other proteins with vesicles, and docking and fusion of vesicles at defined locations. In vertebrates, >75 Rab genes have been identified, some of which have been intensively studied for their roles in endosome and synaptic vesicle trafficking. Recent studies of the functions of certain Rab proteins have revealed specific roles in mediating developmental signal transduction. We have begun a systematic genetic study of the 33 Rab genes in Drosophila. Most of the fly proteins are clearly related to specific vertebrate proteins. We report here the creation of a set of transgenic fly lines that allow spatially and temporally regulated expression of Drosophila Rab proteins. We generated fluorescent protein-tagged wild-type, dominant-negative, and constitutively active forms of 31 Drosophila Rab proteins. We describe Drosophila Rab expression patterns during embryogenesis, the subcellular localization of some Rab proteins, and comparisons of the localization of wild-type, dominant-negative, and constitutively active forms of selected Rab proteins. The high evolutionary conservation and low redundancy of Drosophila Rab proteins make these transgenic lines a useful tool kit for investigating Rab functions in vivo.

Drosophila NMNAT maintains neural integrity independent of its NAD synthesis activity.

Zhai, R.G., Cao, Y., Hiesinger, P.R., Mehta, S.Q., Schulze, K.L., Verstreken, P., Zhou, Y. and Bellen, H.J.

Public Library of Science | 2006-11-28

Appeared In: PLoS Biol. 4(12): e416

Full text [PDF]

Wallerian degeneration refers to a loss of the distal part of an axon after nerve injury. Wallerian degeneration slow (Wld(s)) mice overexpress a chimeric protein containing the NAD synthase NMNAT (nicotinamide mononucleotide adenylyltransferase 1) and exhibit a delay in axonal degeneration. Currently, conflicting evidence raises questions as to whether NMNAT is the protecting factor and whether its enzymatic activity is required for such a possible function. Importantly, the link between nmnat and axon degeneration is at present solely based on overexpression studies of enzymatically active protein. Here we use the visual system of Drosophila as a model system to address these issues. We have isolated the first nmnat mutations in a multicellular organism in a forward genetic screen for synapse malfunction in Drosophila. Loss of nmnat causes a rapid and severe neurodegeneration that can be attenuated by blocking neuronal activity. Furthermore, in vivo neuronal expression of mutated nmnat shows that enzymatically inactive NMNAT protein retains strong neuroprotective effects and rescues the degeneration phenotype caused by loss of nmnat. Our data indicate an NAD-independent requirement of NMNAT for maintaining neuronal integrity that can be exploited to protect neurons from neuronal activity-induced degeneration by overexpression of the protein.

Activity-independent prespecification of synaptic partners in the visual map of Drosophila.

Hiesinger, P.R.* #, Zhai, R.G.*, Zhou, Y., Koh, T.-W., Mehta, S.Q., Verstreken, P., Schulze, K.L., Cao, Y., Fischbach, K.-F., Meinertzhagen, I.A., and Bellen, H.J.#, (2006) *co-first authors, # corresponding authors

Cell Press | 2006-09-19

Appeared In: Curr. Biol. 16(18): 1835-43

Full text [PDF]Suppl. mat. [PDF]

Specifying synaptic partners and regulating synaptic numbers are at least partly activity-dependent processes during visual map formation in all systems investigated to date . In Drosophila, six photoreceptors that view the same point in visual space have to be sorted into synaptic modules called cartridges in order to form a visuotopically correct map . Synapse numbers per photoreceptor terminal and cartridge are both precisely regulated . However, it is unknown whether an activity-dependent mechanism or a genetically encoded developmental program regulates synapse numbers. We performed a large-scale quantitative ultrastructural analysis of photoreceptor synapses in mutants affecting the generation of electrical potentials (norpA, trp;trpl), neurotransmitter release (hdc, syt), vesicle endocytosis (synj), the trafficking of specific guidance molecules during photoreceptor targeting (sec15), a specific guidance receptor required for visual map formation (Dlar), and 57 other novel synaptic mutants affecting 43 genes. Remarkably, in all these mutants, individual photoreceptors form the correct number of synapses per presynaptic terminal independently of cartridge composition. Hence, our data show that each photoreceptor forms a precise and constant number of afferent synapses independently of neuronal activity and partner accuracy. Our data suggest cell-autonomous control of synapse numbers as part of a developmental program of activity-independent steps that lead to a "hard-wired" visual map in the fly brain.

giant fiber A (gfA) encodes a nicotinic acetylcholine receptor that mediates synaptic transmission in the giant fiber circuit of Drosophila

Fayyazuddin, A., Zaheer, M., Hiesinger, P.R., and Bellen, H.J.

Public Library of Science | 2006-02-28

Appeared In: PloS Biology 4(3): e63

Full text [PDF]

Acetylcholine is the major excitatory neurotransmitter in the central nervous system of insects. Mutant analysis of the Dalpha7 nicotinic acetylcholine receptor (nAChR) of Drosophila shows that it is required for the giant fiber-mediated escape behavior. The Dalpha7 protein is enriched in the dendrites of the giant fiber, and electrophysiological analysis of the giant fiber circuit showed that sensory input to the giant fiber is disrupted, as is transmission at an identified cholinergic synapse between the peripherally synapsing interneuron and the dorsal lateral muscle motor neuron. Moreover, we found that gfA1, a mutation identified in a screen for giant fiber defects more than twenty years ago, is an allele of Dalpha7. Therefore, a combination of behavioral, electrophysiological, anatomical, and genetic data indicate an essential role for the Dalpha7 nAChR in giant fiber-mediated escape in Drosophila.

Genetics in the age of systems biology.

Hiesinger, P.R. and Hassan, B.A.

Cell Press | 2005-12-29

Appeared In: Cell 123(7): 1173-4

Full text [PDF]

Systems biology has become a fashionable label for a new generation of large-scale experiments. This essay explores how classical approaches such as forward genetics fit into this emerging framework.

The v-ATPase V0 subunit a1 is required for a late step in synaptic vesicle exocytosis in Drosophila.

cover

Hiesinger, P.R., Fayyazuddin, A., Mehta, S.Q., Rosenmund, T., Schulze, K.L., Zhai, R.G., Verstreken, P., Cao, Y., Zhou, Y., Kunz, J., and Bellen, H.J.

Cell Press | 2005-05-20

Appeared In: Cell 121(4): 607-20

Full text [PDF]

The V(0) complex forms the proteolipid pore of an ATPase that acidifies vesicles. In addition, an independent function in membrane fusion has been proposed largely based on yeast vacuolar fusion experiments. We have isolated mutations in the largest V(0) component vha100-1 in flies in an unbiased genetic screen for synaptic malfunction. The protein is only required in neurons, colocalizes with markers for synaptic vesicles as well as active zones, and interacts with t-SNAREs. Loss of vha100-1 leads to vesicle accumulation in synaptic terminals, suggesting a deficit in release. The amplitude of spontaneous release events and release with hypertonic stimulation indicate normal levels of neurotransmitter loading, yet mutant embryos display severe defects in evoked synaptic transmission and FM1-43 uptake. Our data suggest that Vha100-1 functions downstream of SNAREs in synaptic vesicle fusion.

Mutations in Drosophila sec15 reveal a function in neuronal targeting for a subset of exocyst components.

Mehta, S.Q.*, Hiesinger, P.R.*, Beronja, S., Zhai, R.G., Schulze, K.L., Verstreken, P., Cao, Y., Zhou, Y. Tepass, U., Crair, M.C., and Bellen, H.J. *co-first authors

Cell Press | 2005-04-21

Appeared In: Neuron 46(2): 219-32

Full text [PDF]

The exocyst is a complex of proteins originally identified in yeast that has been implicated in polarized secretion. Components of the exocyst have been implicated in neurite outgrowth, cell polarity, and cell viability. We have isolated an exocyst component, sec15, in a screen for genes required for synaptic specificity. Loss of sec15 causes a targeting defect of photoreceptors that coincides with mislocalization of specific cell adhesion and signaling molecules. Additionally, sec15 mutant neurons fail to localize other exocyst members like Sec5 and Sec8, but not Sec6, to neuronal terminals. However, loss of sec15 does not cause cell lethality in contrast to loss of sec5 or sec6. Our data suggest a role of Sec15 in an exocyst-like subcomplex for the targeting and subcellular distribution of specific proteins. The data also show that functions of other exocyst components persist in the absence of sec15, suggesting that different exocyst components have separable functions.

The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes.

Bellen, H.J., Levis, R.W., Liao, G., He, Y., Carlson, J.W., Tsang, G., Evans-Holm, M., Hiesinger, P.R., Schulze, K.L., Rubin, G.M., Hoskins, R.A., and Spradling, A.C.

Genetics Society Of America | 2004-06

Appeared In: Genetics 167(2): 761-81

http://dx.doi.org/10.1534/genetics.104.0 ...

The Berkeley Drosophila Genome Project (BDGP) strives to disrupt each Drosophila gene by the insertion of a single transposable element. As part of this effort, transposons in >30,000 fly strains were localized and analyzed relative to predicted Drosophila gene structures. Approximately 6300 lines that maximize genomic coverage were selected to be sent to the Bloomington Stock Center for public distribution, bringing the size of the BDGP gene disruption collection to 7140 lines. It now includes individual lines predicted to disrupt 5362 of the 13,666 currently annotated Drosophila genes (39%). Other lines contain an insertion at least 2 kb from others in the collection and likely mutate additional incompletely annotated or uncharacterized genes and chromosomal regulatory elements. The remaining strains contain insertions likely to disrupt alternative gene promoters or to allow gene misexpression. The expanded BDGP gene disruption collection provides a public resource that will facilitate the application of Drosophila genetics to diverse biological problems. Finally, the project reveals new insight into how transposons interact with a eukaryotic genome and helps define optimal strategies for using insertional mutagenesis as a genomic tool.

Flying in the face of total disruption.

Hiesinger, P.R. and Bellen H.J.

Nature Pub. Co. | 2004-03

Appeared In: Nat. Genet. 36(3): 211-2

http://dx.doi.org/10.1038/ng0304-211

More than 50% of the 13,666 Drosophila melanogaster genes are now reported to contain a P-element or piggyBac insertion. Some of these insertions have been used to create molecularly defined deletions spanning more than 50% of the genome.

Visual system development: invertebrates.

Meinertzhagen I.A. and Hiesinger, P.R.

Elesevier | 2004

Appeared In: Elsevier’s Encyclopedia of Neuroscience

Endophilin promotes a late step in endocytosis at glial invaginations in Drosophila photoreceptor terminals

jneuro03-cover

Fabian-Fine, R., Verstreken, P., Hiesinger, P.R., Horne, J.A., Kostyleva, R., Zhou, Y., Bellen, H.J., and Meinertzhagen, I.A.

Society for Neuroscience | 2003-11-19

Appeared In: J. Neurosci. 23, 10732-10744. Cover article

Full text [PDF]

Retrieval of synaptic vesicles from the membrane of neurons is crucial to maintain normal rates of neurotransmitter release. Photoreceptor terminals of the fly's eye release neurotransmitter in a tonic manner. They therefore rely heavily on vesicle regeneration. Null mutations in endophilin (endo) block clathrin-mediated endocytosis at the Drosophila neuromuscular junction, where previous analysis of hypomorphic mutations has suggested a function for Endophilin (Endo) before vesicle fission, during membrane bending. Here, at fly photoreceptor synapses, we show that Endo is localized to synaptic vesicles at sites of endocytosis that are glial invaginations called capitate projections, and that when the photoreceptor synapses lack Endo they are impaired in their ability to release neurotransmitter. Detailed ultrastructural analysis of endo null mutant photoreceptor synapses fails to reveal a defect at early stages of vesicle reformation but, instead, reveals an accumulation of clusters of electron-dense, apparently nonfunctional, late endocytotic vesicles. Using dynamin;endo double-mutant photoreceptors, we provide further evidence that ultimately the function of Endophilin is required late in endocytosis, allowing vesicles to progress through the synaptic vesicle cycle.

Subject: clathrin; endophilin; endocytosis; capitate projection; glia; vesicle cycle

Synaptojanin is recruited by endophilin to promote synaptic vesicle uncoating.

Verstreken, P., Koh, T.-W., Schulze, K.L., Zhai, R.G., Hiesinger, P.R., Zhou, Y., Mehta, S.Q., Cao, Y., Roos, J., and Bellen, H.J.

Cell Press | 2003-11-13

Appeared In: Neuron 40(4): 733-48

http://dx.doi.org/10.1016/S0896-6273(03) ...

We describe the isolation and characterization of Drosophila synaptojanin (synj) mutants. synj encodes a phosphatidylinositol phosphatase involved in clathrin-mediated endocytosis. We show that Synj is specifically localized to presynaptic terminals and is associated with synaptic vesicles. The electrophysiological and ultrastructural defects observed in synj mutants are strikingly similar to those found in endophilin mutants, and Synj and Endo colocalize and interact biochemically. Moreover, synj; endo double mutant synaptic terminals exhibit properties that are very similar to terminals of each single mutant, and overexpression of Endophilin can partially rescue the functional defects in partial loss-of-function synj mutants. Interestingly, Synj is mislocalized and destabilized at synapses devoid of Endophilin, suggesting that Endophilin recruits and stabilizes Synj on newly formed vesicles to promote vesicle uncoating. Our data also provide further evidence that kiss-and-run is able to maintain neurotransmitter release when synapses are not extensively challenged.

Mapping Drosophila mutations with molecularly defined P element insertions.

Zhai, R.G.*, Hiesinger, P.R.*, Koh, T.-W., Verstreken, P., Schulze, K.L., Cao, Y., Jafar-Nejad, H., Norga, K.K., Pan, H., Bayat, V., Greenbaum, M.P., and Bellen, H.J. (2003). *co-first authors

National Academy of Sciences | 2003-09-05

Appeared In: Proc. Natl. Acad. Sci. U.S.A. 100(19): 10860-5

Full text [PDF]

The isolation of chemically induced mutations in forward genetic screens is one of the hallmarks of Drosophila genetics. However, mapping the corresponding loci and identifying the molecular lesions associated with these mutations are often difficult and labor-intensive. Two mapping methods are most often used in flies: meiotic recombination mapping with marked chromosomes and deficiency mapping. The availability of the fly genome sequence allows the establishment and usage of molecular markers. Single-nucleotide polymorphisms have therefore recently been used to map several genes. Here we show that thousands of molecularly mapped P element insertions in fly strains that are publicly available provide a powerful alternative method to single-nucleotide polymorphism mapping. We present a strategy that allows mapping of lethal mutations, as well as viable mutations with visible phenotypes, with minimal resources. The most important unknown in using recombination rates to map at high resolution is how accurately recombination data correlate with molecular maps in small intervals. We therefore surveyed distortions of recombination rates in intervals <500 kb. We document the extent of distortions between the recombination and molecular maps and describe the required steps to map with an accuracy of <50 kb. Finally, we describe a recently developed method to determine molecular lesions in 50-kb intervals by using a heteroduplex DNA mutation detection system. Our data show that this mapping approach is inexpensive, efficient, and precise, and that it significantly broadens the application of P elements in Drosophila.

Shar-pei mediates cell proliferation arrest during imaginal disc growth in Drosophila

devel2002-cover

Kango-Singh, M., Nolo, R., Tao, C., Verstreken, P., Hiesinger, P.R., Bellen, H.J., and Halder, G.

Company Of Biologists Limited | 2002-12

Appeared In: Development 129(24): 5719-5730. Cover article

Full text [PDF]

During animal development, organ size is determined primarily by the amount of cell proliferation, which must be tightly regulated to ensure the generation of properly proportioned organs. However, little is known about the molecular pathways that direct cells to stop proliferating when an organ has attained its proper size. We have identified mutations in a novel gene, shar-pei, that is required for proper termination of cell proliferation during Drosophila imaginal disc development. Clones of shar-pei mutant cells in imaginal discs produce enlarged tissues containing more cells of normal size. We show that this phenotype is the result of both increased cell proliferation and reduced apoptosis. Hence, shar-pei restricts cell proliferation and promotes apoptosis. By contrast, shar-pei is not required for cell differentiation and pattern formation of adult tissue. Shar-pei is also not required for cell cycle exit during terminal differentiation, indicating that the mechanisms directing cell proliferation arrest during organ growth are distinct from those directing cell cycle exit during terminal differentiation. shar-pei encodes a WW-domain-containing protein that has homologs in worms, mice and humans, suggesting that mechanisms of organ growth control are evolutionarily conserved.

Subject: Drosophila, Imaginal discs, Cell proliferation, Apoptosis, WW domain-protein

Drosophila VAP-33A directs bouton formation at neuromuscular junctions in a dosage-dependent manner

neuron2002-cover

Pennetta, G., Hiesinger, P.R., Fabian-Fine, R., Meinertzhagen, I.A., and Bellen H.J.

Cell Press | 2002-07-18

Appeared In: Neuron 35(2): 291-306. Cover article

Full text [PDF]

Aplysia VAP-33 (VAMP-associated protein) has been previously proposed to be involved in the control of neurotransmitter release. Here, we show that a Drosophila homolog of VAP-33, DVAP-33A, is localized to neuromuscular junctions. Loss of DVAP-33A causes a severe decrease in the number of boutons and a corresponding increase in bouton size. Conversely, presynaptic overexpression of DVAP-33A induces an increase in the number of boutons and a decrease in their size. Gain-of-function experiments show that the presynaptic dose of DVAP-33A tightly modulates the number of synaptic boutons. Our data also indicate that the presynaptic microtubule architecture is severely compromised in DVAP-33A mutants. We propose that a DVAP-33A-mediated interaction between microtubules and presynaptic membrane plays a pivotal role during bouton budding.

Drosophila Fragile X protein, DFXR, regulates neuronal morphology and function in the brain.

Morales, J., Hiesinger, P.R., Schroeder, A.J., Kume, K., Verstreken, P., Jackson, F.R., Nelson, D.L., and Hassan, B.A.

Cell Press | 2002-06-13

Appeared In: Neuron 34(6): 961-972

Full text [PDF]

Mental retardation is a pervasive societal problem, 25 times more common than blindness for example. Fragile X syndrome, the most common form of inherited mental retardation, is caused by mutations in the FMR1 gene. Fragile X patients display neurite morphology defects in the brain, suggesting that this may be the basis of their mental retardation. Drosophila contains a single homolog of FMR1, dfxr (also called dfmr1). We analyzed the role of dfxr in neurite development in three distinct neuronal classes. We find that DFXR is required for normal neurite extension, guidance, and branching. dfxr mutants also display strong eclosion failure and circadian rhythm defects. Interestingly, distinct neuronal cell types show different phenotypes, suggesting that dfxr differentially regulates diverse targets in the brain.

Visualization of synaptic markers in the optic neuropils of Drosophila using a new constrained deconvolution method.

Hiesinger, P.R.* , Scholz, M.*, Meinertzhagen, I.A., Fischbach, K.-F., and Obermayer, K. (2001). *co-first and corresponding authors

Wiley-Liss | 2001-01-08

Appeared In: J. Comp. Neurol. 429(2): 277-88

http://dx.doi.org/10.1002/1096-9861(2000 ...

The fruitfly Drosophila melanogaster offers compelling genetic advantages for the analysis of its nervous system, but cell size precludes immunocytochemical analysis of wild-type structure and mutant phenotypes beyond the level of neuronal arborizations. For many antibodies, especially when immunoelectron microscopy is not feasible, it would therefore be desirable to extend the resolution limit of confocal microscopy as far as possible. Because high-resolution confocal microscopy suffers from considerable blurring, so-called deconvolution algorithms are needed to remove, at least partially, the blur introduced by the microscope and by the specimen itself. Here, we present the establishment and application of a new deconvolution method to visualize synaptic markers in Drosophila optic neuropils at the resolution limit of light. We ascertained all necessary parameters experimentally and verified them by deconvolving injected fluorescent microspheres in immunostained optic lobe tissue. The resulting deconvolution method was used to analyze colocalization between the synaptic vesicle marker neuronal synaptobrevin and synaptic and putative synaptic markers in photoreceptor terminals. We report differential localization of these near the resolution limit of light, which could not be distinguished without deconvolution.

Subject: visual system; photoreceptor; synaptobrevin; syntaxin; cysteine string protein; IrreC-rst;confocal microscopy

The evolution of the millenium bug

Hiesinger, P.R. and Bellen H.J.

Tilgher-Genova | 2000

Appeared In: Riv Biol. 93(1): 169-74

How could mankind, knowing the year 2000 would inevitably arrive, manoeuvre into worldwide technical problems because of a little computer bug? Two major parallels can be drawn to biological systems, and both are based on evolutionary principles. First, any new steps in development are founded on building blocks invented earlier. Basic building blocks are hardly changed anymore because further developments depend on their function. Second, imperfections of such building blocks are irrelevant as long as no corresponding selection pressure exists. If a time-coded computer bug occurs sufficiently early during technological development it can become part of innumerous hard-wired or soft-coded programs and devices without ever attracting attention. However, the arrival of a certain data can instantly put a high selection pressure upon it. This behaviour can be understood as a direct consequence of the autonomous dynamics that the development of complex systems implicates.

Neuropil pattern formation and regulation of cell adhesion molecules in Drosophila optic lobe development depend on synaptobrevin.

jneuro99-cover

Hiesinger, P.R., Reiter, C., Schau, H., and Fischbach, K.-F.

Society for Neuroscience | 1999-09-01

Appeared In: J. Neurosci. 19(17): 7548-56

Full text [PDF]

To investigate a possible involvement of synaptic machinery in Drosophila visual system development, we studied the effects of a loss of function of neuronal synaptobrevin, a protein required for synaptic vesicle release. Expression of tetanus toxin light chain (which cleaves neuronal synaptobrevin) and genetic mosaics were used to analyze neuropil pattern formation and levels of selected neural adhesion molecules in the optic lobe. We show that targeted toxin expression in the developing optic lobe results in disturbances of the columnar organization of visual neuropils and of photoreceptor terminal morphology. IrreC-rst immunoreactivity in neuropils is increased after widespread expression of toxin. In photoreceptors, targeted toxin expression results in increased Fasciclin II and chaoptin but not IrreC-rst immunoreactivity. Axonal pathfinding and programmed cell death are not affected. In genetic mosaics, patches of photoreceptors that lack neuronal synaptobrevin exhibit the same phenotypes observed after photoreceptor-specific toxin expression. Our results demonstrate the requirement of neuronal synaptobrevin for regulation of cell adhesion molecules and development of the fine structure of the optic lobe. A possible causal link to fine-tuning processes that may include synaptic plasticity in the development of the Drosophila CNS is discussed.

Subject: Drosophila, optic lobe development, synaptobrevin, synaptic plasticity, cell adhesion molecules, tetanus toxin, Fasciclin II, IrreC-rst, chaoptin

Three-dimensional reconstruction of the antennal lobe in Drosophila melanogaster.

Laissue, P.P., Reiter, C., Hiesinger, P.R., Halter, S., Fischbach, K.-F., and Stocker, R.F.

Wiley-Liss | 1999-03-22

Appeared In: J. Comp. Neurol. 405(4): 543-52

http://dx.doi.org/10.1002/(SICI)1096-986 ...

We present the first three-dimensional map of the antennal lobe of Drosophila melanogaster, based on confocal microscopic analysis of glomeruli stained with the neuropil-specific monoclonal antibody nc82. The analysis of confocal stacks allowed us to identify glomeruli according to the criteria shape, size, position, and intensity of antibody labeling. Forty glomeruli were labeled by nc82, eight of which have not been described before. Three glomeruli previously shown exclusively by backfills were not discernible in nc82 stainings. We distinguish three classes of glomeruli: (1) "landmark" glomeruli that are constant in all four criteria mentioned above, (2) less well-demarcated glomeruli that deviate in a single criterion, and (3) poorly defined glomeruli that vary in more than one criterion. All class 2 and 3 glomeruli can be identified by comparison with landmark neighbors. To further aid identification, our model assigns glomeruli to five arrays, each of which is defined by a prominent landmark glomerulus. Six glomeruli consist of distinct, but contiguous structural units, termed "compartments." Glomerular variability observed occasionally between males and females is in the same range as between individuals of the same sex, suggesting the lack of a significant sexual dimorphism in the glomerular pattern. We compare the new model with a previous map and address its potential for mapping activity and expression patterns. An important goal of this work was to create three-dimensional reference models of the antennal lobe, which are accessible on-line.

Subject: olfactory system; glomeruli; confocal microscopy; enhancer trap lines; rendering; VRML