Steven J Husson et al. (2009) European Worm Neurobiology Meeting "Optogenetics tools to dissect a nociceptive neuronal network and neuropeptide signalling pathways in C. elegans"

Alexander Gottschalk, Jana Liewald, Steven J Husson, Liliane Schoofs, Martin Brauner, Wagner Steuer Costa

[European Worm Neurobiology Meeting, 2009]

Light-gated cation channels such as the blue light-activated depolarizing Channelrhodopsin-2 (ChR2), allow optical activation of individual neurons of live and behaving C. elegans at the millisecond time-scale in a non-invasive manner (Nagel et al., 2005, Zhang et al., 2007, Liewald et al., 2008). This optogenetics approach paves the way for further functional dissection of peptide signalling pathways or individual neuronal networks in a detail that is not possible in higher organisms. The huge advantage is that we can specifically stimulate the sensory input neurons, while other potentially contributing neurons are kept silent. The sensory PVD neurons that envelop the nematode with highly branched dendritic arbors are involved in harsh touch nociception. Expression and activation of ChR2 in PVD results in a forward escape movement and sometimes a reversal. These results are in line with the fact that the PVD neurons make synaptic contacts with the locomotory command interneurons PVC and AVA that regulate forward and backward movement, respectively. Using electrophysiology, we will assess the physiology of the PVD cells, as well as the downstream interneurons in response to photoactivation, while the involvement of different ion channels, receptors or neurotransmitters will be assessed by RNAi. We are also investigating the flp-15 and nlp-38 neuropeptidergic signalling pathways by optogenetics. Neuropeptide release can be triggered by photo-activating the respective neurons in an acute fashion while effects on behaviour can be observed at the same time. This way, we can correlate neuropeptide action with acute behavioural changes or effects, about which very limited knowledge is currently available in any system. References Liewald JF, Brauner M, Stephens GJ, Bouhours M, Schultheis C, Zhen M, and Gottschalk A (2008) Optogenetic analysis of synaptic function. Nat Methods, 5, 895-902. Nagel G, Brauner M, Liewald JF, Adeishvili N, Bamberg E, and Gottschalk A (2005) Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr Biol, 15, 2279-2284. Zhang F, Wang LP, Brauner M, Liewald JF, Kay K, Watzke N, Wood PG, Bamberg E, Nagel G, Gottschalk A, and Deisseroth K (2007) Multimodal fast optical interrogation of neural circuitry. Nature, 446, 633-639.

Husson, Steven J et al. (2009) International Worm Meeting "Optogenetics tools to dissect small neuronal networks and neuropeptide signalling systems."

Liewald, Jana, Husson, Steven J, Schultheis, Christian, Schoofs, Liliane, Gottschalk, Alexander, Brauner, Martin, Erbguth, Karen, Schedletzky, Thorsten

[International Worm Meeting, 2009]

Light-gated ion channels or pumps such as the blue light-activated depolarizing Channelrhodopsin (ChR2) and the yellow light-driven hyperpolarizing Halorhodopsin (HR) allow optical activation or inhibition in muscles and neurons of live and behaving C. elegans (Zhang et al., 2007). Furthermore, inward currents evoked by either ChR2 or HR, as well as muscle currents in response to activating ChR2 in motor neurons, can be directly measured by electrophysiology, while photo-evoked body contraction or elongation of the animal could be monitored at the behavioural level (Nagel et al., 2005; Liewald et al., 2008). These state-of-the-art technologies pave the way for further functional dissection of individual neuronal networks in a detail that is not possible in higher organisms. Doing so, we are investigating some defined neuropeptidergic signalling pathways. Neuropeptide release can be triggered by photo-activating the respective neurons in an acute fashion while effects on behaviour can be observed at the same time. This way, we can correlate neuropeptide action with acute behavioural changes or effects, about which very limited knowledge is currently available in any system. While higher organisms display millions of contributing neurons, only a handful of neurons take part in individual neuronal networks in C. elegans. This opens the possibility to study the contribution of each neuron to the function of a small network, for example involved in nociception. The huge advantage of our optogenetics tools is that we can specifically stimulate the sensory input neurons, while other potentially contributing neurons are kept silent. The involvement of different ion channels, receptors or neurotransmitters can be assessed by using different genetic backgrounds, while the physiological properties of each individual neuron will be monitored by electrophysiology. Liewald JF, Brauner M, Stephens GJ, Bouhours M, Schultheis C, Zhen M, and Gottschalk A (2008) Optogenetic analysis of synaptic function. Nat Methods, 5, 895-902. Nagel G, Brauner M, Liewald JF, Adeishvili N, Bamberg E, and Gottschalk A (2005) Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr Biol, 15, 2279-2284. Zhang F, Wang LP, Brauner M, Liewald JF, Kay K, Watzke N, Wood PG, Bamberg E, Nagel G, Gottschalk A, and Deisseroth K (2007) Multimodal fast optical interrogation of neural circuitry. Nature, 446, 633-639.

Ellen Nollen et al. (2003) International Worm Meeting "Genome-wide RNAi screen for genes involved in polyglutamine aggregate formation"

Ronald H Plasterk, Richard I Morimoto, Gijs van Haaften, Susana Garcia, Ellen Nollen

[International Worm Meeting, 2003]

Accumulation of polyglutamine containing proteins into intracellular aggregates is associated with various CAG trinucleotide expansion disorders, including neurodegenerative diseases such as Huntingtons disease and spinocerebellar ataxias. The aggregation properties of polyglutamine proteins are directly related to the length of the polyglutamine stretch. With polyglutamine stretches above a threshold of approximately 30 glutamine residues, the aggregation rate increases with increasing numbers of glutamine residues. The length-dependent kinetics of aggregation recapitulates the length-dependent increase in cellular toxicity and age of onset of disease. Expression of polyglutamine stretches of 0, 24, 33, 35, 40, 44, and 82 glutamine residues as YFP-fusion proteins under control of the muscle specific unc-54 promoter in C. elegans reconstitutes the length and age dependence of aggregation. 1 Whereas worms expressing YFP-fusions with polyglutamine stretches up to 24 (Q24) show a diffuse YFP staining in all muscle cells, Q82 animals show a punctate staining in most of the cells. Interestingly, all Q lengths show variability in aggregation within individual animals, depending on the cell and the age of the worm, which is influenced by the genetic background of the worms. For example, aggregation of Q82-YFP is greatly delayed in the aging mutant age-11. This heterogeneity of aggregation suggests that genes exist that influence the formation of polyglutamine aggregates. We are using a genome-wide RNAi screen to identify genes involved in polyglutamine aggregation. In a candidate gene approach with RNAi against genes encoding molecular chaperones and molecules involved in proteins degradation, we have already identified genes that may play a role in aggregate formation. 1Morley JF, Brignull HR, Weyers JJ, Morimoto RI. Proc Natl Acad Sci U S A 2002 99(16):10417-22

Anusha Narayan et al. (2007) International Worm Meeting "Transfer at C. elegans synapses."

Shawn Lockery, Paul Sternberg, Tod Thiele, Gilles Laurent, Anusha Narayan

[International Worm Meeting, 2007]

Neural circuits in C. elegans have been studied using light and electron microscopic techniques, focal laser ablations and, more recently, calcium imaging techniques. For a clearer functional understanding of these circuits, however, some knowledge of the rules of synaptic information transfer is required. How is the dynamic range of the post-synaptic neuron set? What are the mechanisms for synaptic integration and gain control? Questions such as these can best be answered by monitoring or controlling connected pre- and post-synaptic neurons simultaneously. We chose to focus on the synapses between the AFD/ASER and AIY neurons, since the functional relevance of these neurons has been established and there is anatomical evidence for synapses between them. Channelrhodopsin-2 (chR2) is a light activated cation channel with fast kinetics (order of milliseconds1). We express chR2 under a neuron-specific promoter2 in the presynaptic neuron, and use whole-cell patch-clamp recording techniques to monitor membrane voltage or currents in the postsynaptic neuron. We are first calibrating the response to light of chR2-expressing neurons. Currently, we are calibrating this light response in worms expressing chR2 in ASER. We have observed depolarizations of 10-30 mV in response to light (450-490 nm) in current clamp, and inward currents of 5-10 pA in voltage-clamp. We have also seen evidence of spontaneous synaptic activity, in the form of discrete synaptic events (potentials or currents) with different reversal potentials (some depolarizing, others Cl-dependent). We are beginning to characterize the ASER-AIY synapse, and will then move on to the AFD-AIY synapse. References 1. Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K. Millisecond-timescale, genetically targeted optical control of neural activity, Nat. Neurosci. 8 (2005), pp. 1263-1268. 2. Nagel G, Brauner M, Liewald JF, Adeishvili N, Bamberg E, Gottschalk A. Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr. Biol. 15 (2005), pp. 2279-2284.

Nehal Mehta et al. (2002) West Coast Worm Meeting "Isolation and Characterization of Genes Involved in Ectopic Neurite Outgrowth Defects"

Oliver Hobert, Paula Loria, Nehal Mehta

[West Coast Worm Meeting, 2002]

Blockade of communication between neurons or between neurons and muscle has been shown to cause ectopic axonal outgrowth in vertebrates and more recently in C. elegans (Holland and Brown 1980, Nonet et al. 2000). The ectopic axonal outgrowth or sprouting phenotype indicates that neurons are able to monitor and make compensatory attempts to maintain functional innervation of the target. Such activity-dependent remodeling may be caused by retrograde signaling from the target to the neuron leading to cytoskeletal rearrangements. In order to understand the nature of neuromuscular communication, we have chosen the DVB neuron as our model system. DVB is a postembryonically born GABAergic motor neuron that mediates the expulsion step of the defecation motor program. Disruptions in neural differentiation (lim-6), activity (unc-25, unc-104), and axogenesis (unc-44, unc-34) lead to ectopic outgrowth defects in DVB. Furthermore, silencing of the enteric muscle by egl-2(gf), a hyperactivated eag-like K+ channel, or lack of enteric muscle formation by hlh-8, the Twist homolog, also causes DVB ectopic outgrowth defects. This suggests that neuronal innervation and activation of muscle may promote the feedback of a retrograde signal to prevent further axonal outgrowth. To decipher the mechanisms behind this bidirectional neuromuscular communication, we have conducted a screen for mutants that display ectopic neurite outgrowth defects (the eno phenotype) in DVB. Isolated mutants can represent genes involved in DVB functional development, maturation, or maintenance. A transgenic strain expressing unc47::gfp (oxIs12) (McIntire et al., 1997) was mutagenized using ethyl methanesulfate (EMS) and the progeny of almost 3000 mutagenized F1 animals were screened using a compound microscope for the eno phenotype. 12 eno mutants have been retrieved and placed into 11 complementation groups. We have started to clone and characterize eno-1. In addition to DVB sprouting, eno-1 also shows ectopic outgrowth defects in the AVL motor neuron, which has redundant functions with DVB in mediating the expulsion step of defecation. Eno-1 has a stong hypersensitive phenotype on aldicarb, suggesting that it may be involved in negative regulation of neurotransmission. Eno-1 has been mapped between lin-31 and rol-6 on chromosome II. By snip-SNP mapping, eno-1 has been narrowed down to 9 cosmids. Further mapping results will be presented.

George SE et al. (1998) West Coast Worm Meeting "SEARCHING FOR COMPONENTS OF VAB-1 PATHWAYS"

Pham T, George SE, Chisholm AD, Grinberg I

[West Coast Worm Meeting, 1998]

vab-1 mutants are defective in many aspects of neural and epidermal morphogenesis, including ventral epidermal enclosure during embryogenesis and in neuroblast migrations following gastrulation. vab-1 encodes an Eph Receptor Tyrosine Kinase (RTK) (George et al., Cell 92: 633, 1998). Eph receptors are the largest subfamily of vertebrate RTK and are important in directing axonal guidance and morphogenesis in vertebrates. Based on the expression pattern of VAB-1::GFP constructs and genetic mosaic analysis, VAB-1 may function in the nervous system during embryonic morphogenesis. We are further examining where VAB-1 function is necessary and how VAB-1 signaling affects epidermal and neural development. Our previous genetic mosaic analysis suggested that VAB-1 functions in neural cells for normal epidermal morphogenesis. A caveat to this genetic mosaic analysis was that embryonic phenotypes were not assessed. A vab-1 cDNA expressed under the control of the pan-neural unc-119 promoter can rescue the embryonic phenotypes of vab-1 null mutants. Thus, vab-1 expression in neurons is sufficient for normal epidermal development. We have also created a dominant-negative (DN) version of VAB-1 by deleting the intracellular domain of the receptor. Animals that express this VAB-1DN construct under the control of the unc-119 promoter display embryonic mutant phenotypes similar to those of strong vab-1 alleles. If this VAB-1DN construct acts via inhibition of normal VAB-1 signaling pathways, then vab-1 function in neurons may be necessary for normal embryonic morphogenesis. To further understand how VAB-1 signaling controls morphogenesis, we are seeking additional components of the VAB-1 signal transduction pathway. Eph RTKs are activated by binding Ephrin ligands. Several proteins that bind to activated Eph receptors have been identified by biochemical approaches, such as the adaptor protein Nck (Holland et al., EMBO J. 16: 3877, 1997); however, the roles of such proteins in vivo are poorly understood. We are testing whether C. elegans homologs of such proteins interact with VAB-1 using the yeast two-hybrid system. We are also using the two-hybrid system to screen for new VAB-1-binding proteins. We are also designing genetic screens to identify genes that act downstream of the VAB-1 kinase. We have constructed a constitutively activated version of the VAB-1 receptor by replacing the extracellular ligand-binding domain with the lambda repressor dimerization domain. This should cause ligand-independent dimerization and constitutive activation of VAB-1 kinase-dependent pathways. We are testing various promoters to drive this construct to produce a phenotype with which we will be able to screen for downstream effectors of Eph signal transduction.

Hidehito KUROYANAGI et al. (2001) International C. elegans Meeting "C. elegans SR protein kinase, SPK-1, is required for germline proliferation and maturation."

Hidehito KUROYANAGI, Masatoshi Hagiwara

[International C. elegans Meeting, 2001]

SR-protein kinases (SRPKs) and their substrates, the serine/arginine-rich pre-mRNA splicing factors (SR proteins), are key components of splicing machinery and are well conserved across phyla. Despite extensive biochemical investigation, the physiological functions of SRPKs remain unclear. In C. elegans , one SRPK gene and six SR protein genes have been reported. Functional analyses by us and other labs utilizing RNAi technique revealed that one member of SR protein family, CeSF2/ASF, is essential for embryogenesis (1,2), while other SR proteins play redundant roles (2,3); spk-1 is required for early embryogenesis (1,2). However, we have also demonstrated that spk-1 is required in germline proliferation and gametogenesis utilizing various RNAi protocols (1). Here we report the isolation and analyses of a deletion mutant within the spk-1 gene. spk-1 mutants are comparable to Wild-Type in locomotion and body size. However, spk-1 mutants shows recessive fully penetrated phenotype of sterility in both hermaphrodites and males. In spk-1 hermaphrodites germlines are underproliferated and lack oogenesis. spk-1 males have comparable number of germ nuclei in a gonadal arm, suggesting deficiency in sperm maturation. These observations indicate that zygotic expression of spk-1 is required for germline proliferation in hermaphrodites and maturation in both hermaphrodites and males. In order to analyze the epistasis between germline proliferation deficiency in spk-1 mutant and ectopic or tumorous proliferation of germline in gld-2 (h292) and gld-1; gld-2 double mutant, we generated double and triple mutants. Certain population of gld-2 (h292); spk-1 showed ectopic proliferation of germ nuclei in the most proximal region of the gonad, indicating that gld-2 (h292) is epistatic. On the other hand, gld-2 (q497) gld-1 (q485); spk-1 showed Glp phenotype, indicating that spk-1 is epistatic. With these characteristics, spk-1 mutant may serve as a useful tool to analyze the molecular mechanisms in sex-determination, proliferation and maturation of C. elegans germline. 1: Kuroyanagi H, Kimura T, Wada K, Hisamoto N, Matsumoto K, Hagiwara M. SPK-1, a C. elegans SR protein kinase homologue, is essential for embryogenesis and required for germline development. Mech Dev. 2000; 99(1-2): 51-64. 2: Longman D, Johnstone IL, Caceres JF.@ Functional characterization of SR and SR-related genes in Caenorhabditis elegans . EMBO J. 2000; 19(7): 1625-37. 3: Kawano T, Fujita M, Sakamoto H. Unique and redundant functions of SR proteins, a conserved family of splicing factors, in Caenorhabditis elegans development. Mech Dev. 2000; 95(1-2): 67-76.

Nehal Mehta et al. (2001) International C. elegans Meeting "Isolation and Characterization of Genes Involved in Motor Axon Sprouting"

Nehal Mehta, Paula Loria, Oliver Hobert

[International C. elegans Meeting, 2001]

Disruption of neuron-target communication has been shown to cause axonal sprouting in vertebrates and more recently in C. elegans (Holland and Brown, 1980; Nonet et al., 2000; Peckol et al., 1999). The neuronal sprouting defect may represent an attempt to reestablish a functional synapse. Activity dependent remodeling is presumably caused by retrograde signaling from the target to the neuron leading to cytoskeletal rearrangement. However, the nature of retrograde signals remains elusive. In an attempt to identify a potential retrograde signal at the neuromuscular junction, we have decided to study the DVB motor neuron. DVB is a postembryonically born GABAergic neuron that mediates the expulsion step of the defecation motor program by supplying synaptic output onto the enteric and anal depressor muscles. A number of known mutants involved in neuron differentiation or synaptic activity have been tested for DVB sprouting. A modification in the transcriptional control program for motor neuron differentiation due to a mutation in the Lim homeobox transcription factor, lim-6 , reveals DVB neurite sprouting (Hobert et al., 1999). Disruption of anterograde signaling by the kinesin gene unc-104 or by the GABA synthesizing enzyme unc-25 also displays DVB sprouting. Axogenesis mutations in the ankyrin gene unc-44 , the septin gene unc-61 , or the enabled homologue unc-34 cause DVB sprouting. Blockade of enteric muscle activity by hyperactivation of the eag -like K + channel egl-2 (gf) or loss of enteric muscle formation in hlh-8 mutants manifests DVB motor neuron sprouting. Silencing or absence of the target muscle reveals that the DVB neuron must have an intrinsic propensity to sprout. Having established that neural activity dependent communication is required to suppress neurite sprouting, we have conducted a screen for DVB motor neuron sprouting with the goal of identifying genes that are specifically involved in retrograde signaling. Such a screen may also yield anterograde signaling and lim-6 interacting components. A transgenic strain expressing unc-47 ::GFP ( oxIs12 ) (McIntire et al., 1997) was mutagenized using ethyl methanesulfate (EMS) and the progeny of almost 3000 mutagenized F 1 animals were screened using a compound microscope for DVB motor neuron sprouting. Twelve mutants, ot1-ot9 , ot32 , ot40 , and ot42 have been isolated and placed into at least 5 different complementation groups. All of these mutant animals show a sprouting defect of at least 30%. No obvious pleiotropies such as body morphology and locomotion are affected suggesting that these mutants may represent different or even novel genes than known synaptic activity mutants which tend to display an uncoordinated phenotype. Visualization of ventral and dorsal nerve cords and D-type motor neurons using unc-47 ::GFP, the AIY interneuron using ttx-3 ::GFP, and sensory neurons using DiI filling reveals that ot1 - ot9 appear to specifically affect DVB. Ot42 , however, displays pleiotropic neuron sprouting defects comparable to sensory axon defective (sax) mutants and has shown to complement sax-1 , sax-2 , and sax-6 (Zallen et al., 1999). The presence of the enteric muscle using the transgenic strain ceh-24Nde ::GFP ( nuIs63 ) (Madison and Kaplan, pers. comm.) has been confirmed for ot1-ot9 . I have started cloning and characterizing ot1 , ot6 , and ot42 . Three factor mapping has placed ot1 between lin-31 and rol-6 on chromosome II. Further mapping of single nucleotide polymorphisms (SNPs) between the N2 and the Hawaiin strains will narrow down a region for rescue. Also by three factor mapping, ot6 has been placed between bp1 and sma-1 on chromosome V. Candidate genes are being amplified and sequenced and cosmids will be injected for rescue. Further cloning and characterization of these three mutants will elucidate potential roles in motor neuron differentiation, anterograde activity, and retrograde signaling.