Kaushiki P. Menon

A gain-of-function screen for genes controlling motor axon guidance and synaptogenesis in Drosophila.

BACKGROUND The neuromuscular system of the Drosophila larva contains a small number of identified motor neurons that make genetically defined synaptic connections with muscle fibers. We drove high-level expression of genes in these motor neurons by crossing 2293 GAL4-driven EP element lines with known insertion site sequences to lines containing a pan-neuronal GAL4 source and UAS-green fluorescent protein elements. This allowed visualization of every synapse in the neuromuscular system in live larvae. RESULTS We identified 114 EPs that generate axon guidance and/or synaptogenesis phenotypes in F1 EP x driver larvae. Analysis of genomic regions adjacent to these EPs defined 76 genes that exhibit neuromuscular gain-of-function phenotypes. Forty-one of these (known genes) have published mutant alleles; the other 35 (new genes) have not yet been characterized genetically. To assess the roles of the known genes, we surveyed published data on their phenotypes and expression patterns. We also examined loss-of-function mutants ourselves, identifying new guidance and synaptogenesis phenotypes for eight genes. At least three quarters of the known genes are important for nervous system development and/or function in wild-type flies. CONCLUSIONS Known genes, new genes, and a set of previously analyzed genes with phenotypes in the Adh region display similar patterns of homology to sequences in other species and have equivalent EST representations. We infer from these results that most new genes will also have nervous system loss-of-function phenotypes. The proteins encoded by the 76 identified genes include GTPase regulators, vesicle trafficking proteins, kinases, and RNA binding proteins.

A Screen of Cell-Surface Molecules Identifies Leucine-Rich Repeat Proteins as Key Mediators of Synaptic Target Selection

In Drosophila embryos and larvae, a small number of identified motor neurons innervate body wall muscles in a highly stereotyped pattern. Although genetic screens have identified many proteins that are required for axon guidance and synaptogenesis in this system, little is known about the mechanisms by which muscle fibers are defined as targets for specific motor axons. To identify potential target labels, we screened 410 genes encoding cell-surface and secreted proteins, searching for those whose overexpression on all muscle fibers causes motor axons to make targeting errors. Thirty such genes were identified, and a number of these were members of a large gene family encoding proteins whose extracellular domains contain leucine-rich repeat (LRR) sequences, which are protein interaction modules. By manipulating gene expression in muscle 12, we showed that four LRR proteins participate in the selection of this muscle as the appropriate synaptic target for the RP5 motor neuron.

Development and plasticity of the Drosophila larval neuromuscular junction

The Drosophila larval neuromuscular system is relatively simple, containing only 32 motor neurons in each abdominal hemisegment, and its neuromuscular junctions (NMJs) have been studied extensively. NMJ synapses exhibit developmental and functional plasticity while displaying stereotyped connectivity. Drosophila Type I NMJ synapses are glutamatergic, while the vertebrate NMJ uses acetylcholine as its primary neurotransmitter. The larval NMJ synapses use ionotropic glutamate receptors (GluRs) that are homologous to AMPA‐type GluRs in the mammalian brain, and they have postsynaptic scaffolds that resemble those found in mammalian postsynaptic densities. These features make the Drosophila neuromuscular system an excellent genetic model for the study of excitatory synapses in the mammalian central nervous system. The first section of the review presents an overview of NMJ development. The second section describes genes that regulate NMJ development, including: (1) genes that positively and negatively regulate growth of the NMJ, (2) genes required for maintenance of NMJ bouton structure, (3) genes that modulate neuronal activity and alter NMJ growth, (4) genes involved in transsynaptic signaling at the NMJ. The third section describes genes that regulate acute plasticity, focusing on translational regulatory mechanisms. As this review is intended for a developmental biology audience, it does not cover NMJ electrophysiology in detail, and does not review genes for which mutations produce only electrophysiological but no structural phenotypes. WIREs Dev Biol 2013, 2:647–670. doi: 10.1002/wdev.108

The Translational Repressors Nanos and Pumilio Have Divergent Effects on Presynaptic Terminal Growth and Postsynaptic Glutamate Receptor Subunit Composition

Pumilio (Pum) is a translational repressor that binds selectively to target mRNAs and recruits Nanos (Nos) as a corepressor. In the larval neuromuscular system, Pum represses expression of the translation factor eIF-4E and the glutamate receptor subunit GluRIIA. Here, we show that Nos, like Pum, is expressed at the neuromuscular junction (NMJ) and in neuronal cell bodies. Surprisingly, however, Nos and Pum have divergent functions on both the presynaptic and postsynaptic sides of the NMJ. In nos mutant and nos RNA interference larvae, the number of NMJ boutons is increased, whereas loss of Pum reduces the bouton number. On the postsynaptic side, Nos acts in opposition to Pum in regulating the subunit composition of the glutamate receptor. NMJ active zones are associated with GluRIIA- and GluRIIB-containing receptor clusters. Loss of Nos causes downregulation of GluRIIA and increases the levels of GluRIIB. Consistent with this finding, the electrophysiological properties of NMJs lacking postsynaptic Nos suggest that they use primarily GluRIIB-containing receptors. Nos can regulate GluRIIB in the absence of GluRIIA, suggesting that the effects of Nos on GluRIIB levels are at least partially independent of synaptic competition between GluRIIA and GluRIIB. Nos is a target for Pum repression, and Pum binds selectively to the 3′ untranslated regions of the nos and GluRIIA mRNAs. Our results suggest a model in which regulatory interplay among Pum, Nos, GluRIIA, and GluRIIB could cause a small change in Pum activity to be amplified into a large shift in the balance between GluRIIA and GluRIIB synapses.

Control of Synaptic Connectivity by a Network of Drosophila IgSF Cell Surface Proteins

We have defined a network of interacting Drosophila cell surface proteins in which a 21-member IgSF subfamily, the Dprs, binds to a nine-member subfamily, the DIPs. The structural basis of the Dpr-DIP interaction code appears to be dictated by shape complementarity within the Dpr-DIP binding interface. Each of the six dpr and DIP genes examined here is expressed by a unique subset of larval and pupal neurons. In the neuromuscular system, interactions between Dpr11 and DIP-γ affect presynaptic terminal development, trophic factor responses, and neurotransmission. In the visual system, dpr11 is selectively expressed by R7 photoreceptors that use Rh4 opsin (yR7s). Their primary synaptic targets, Dm8 amacrine neurons, express DIP-γ. In dpr11 or DIP-γ mutants, yR7 terminals extend beyond their normal termination zones in layer M6 of the medulla. DIP-γ is also required for Dm8 survival or differentiation. Our findings suggest that Dpr-DIP interactions are important determinants of synaptic connectivity.

Immunolocalization of synaptotagmin for the study of synapses in the developing antennal lobe of Manduca sexta

In the mature olfactory systems of most organisms that possess a sense of smell, synapses between olfactory receptor neurons and central neurons occur in specialized neuropil structures called glomeruli. The development of olfactory glomeruli has been studied particularly heavily in the antennal lobe of the moth Manduca sexta. In the current study, we address the development of synapses within the antennal lobe of M. sexta by reporting on the localization of synaptotagmin, a ubiquitous synaptic vesicle protein, throughout development. A cDNA clone coding for M. sexta synaptotagmin was characterized and found to encode a protein that shares 67% amino acid identity with Drosophila synaptotagmin and 56% amino acid identity with human synaptotagmin I. Conservation was especially high in the C2 domains near the C‐terminus and very low near the N‐terminus. A polyclonal antiserum (MSYT) was raised against the unique N‐terminus of M. sexta synaptotagmin, and a monoclonal antibody (DSYT) was raised against the highly conserved C‐terminus of D. melanogaster synaptotagmin. In Western blot analyses, both antibodies labeled a 60 kD protein, which very likely corresponds to synaptotagmin. On sections, both antibodies labeled known synaptic neuropils in M. sexta and yielded similar labeling patterns in the developing antennal lobe. In addition, DSYT detected synaptotagmin‐like protein in three other insect species examined. Analysis of synaptotagmin labeling at the light microscopic level during development of the antennal lobe of M. sexta confirmed and extended previous electron microscopic studies. Additional synapses in the coarse neuropil and a refinement of synaptic densities in the glomeruli during the last one‐third of metamorphic development were revealed. J. Comp. Neurol. 441:277–287, 2001.

Regulation of Synaptic Pumilio Function by an Aggregation-Prone Domain

We identified Pumilio (Pum), a Drosophila translational repressor, in a computational search for metazoan proteins whose activities might be regulated by assembly into ordered aggregates. The search algorithm was based on evolutionary sequence conservation patterns observed for yeast prion proteins, which contain aggregation-prone glutamine/asparagine (Q/N)-rich domains attached to functional domains of normal amino acid composition. We examined aggregation of Pum and its nematode ortholog PUF-9 by expression in yeast. A domain of Pum containing the Q/N-rich sequence, denoted as NQ1, the entire Pum N terminus, and the complete PUF-9 protein localize to macroscopic aggregates (foci) in yeast. NQ1 and PUF-9 can generate the yeast Pin+ trait, which is transmitted by a heritable aggregate. NQ1 also assembles into amyloid fibrils in vitro. In Drosophila, Pum regulates postsynaptic translation at neuromuscular junctions (NMJs). To assess whether NQ1 affects synaptic Pum activity in vivo, we expressed it in muscles. We found that it negatively regulates endogenous Pum, producing gene dosage-dependent pum loss-of-function NMJ phenotypes. NQ1 coexpression also suppresses lethality and NMJ phenotypes caused by overexpression of Pum in muscles. The Q/N block of NQ1 is required for these phenotypic effects. Negative regulation of Pum by NQ1 might be explained by formation of inactive aggregates, but we have been unable to demonstrate that NQ1 aggregates in Drosophila. NQ1 could also regulate Pum by a “dominant-negative” effect, in which it would block Q/N-mediated interactions of Pum with itself or with cofactors required for translational repression.

The translational regulator Cup controls NMJ presynaptic terminal morphology

During oogenesis and early embryonic development in Drosophila, translation of proteins from maternally deposited mRNAs is tightly controlled. We and others have previously shown that translational regulatory proteins that function during oogenesis also have essential roles in the nervous system. Here we examine the role of Cup in neuromuscular system development. Maternal Cup controls translation of localized mRNAs encoding the Oskar and Nanos proteins and binds to the general translation initiation factor eIF4E. In this paper, we show that zygotic Cup protein is localized to presynaptic terminals at larval neuromuscular junctions (NMJs). cup mutant NMJs have strong phenotypes characterized by the presence of small clustered boutons called satellite boutons. They also exhibit an increase in the frequency of spontaneous glutamate release events (mEPSPs). Reduction of eIF4E expression synergizes with partial loss of Cup expression to produce satellite bouton phenotypes. The presence of satellite boutons is often associated with increases in retrograde bone morphogenetic protein (BMP) signaling, and we show that synaptic BMP signaling is elevated in cup mutants. cup genetically interacts with two genes, EndoA and Dap160, that encode proteins involved in endocytosis that are also neuronal modulators of the BMP pathway. Endophilin protein, encoded by the EndoA gene, is downregulated in a cup mutant. Our results are consistent with a model in which Cup and eIF4E work together to ensure efficient localization and translation of endocytosis proteins in motor neurons and control the strength of the retrograde BMP signal.