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.

Sym M et al. (2000) West Coast Worm Meeting "Two new genes regulating neuroblast migration in C. elegans"

Kenyon CJ, Yang L, Sym M, Ch'ng Q-L

[West Coast Worm Meeting, 2000]

In C. elegans, the Q cells are bilaterally symmetric neuroblasts present in the posterior body region of the worm at hatching. During the first larval stage, the Q cells divide and migrate. QR and its descendents migrate anteriorly whereas QL and its descendents migrate posteriorly. Several genes that regulate the anterior migrations of QR and its descendents have been identified. These include: 1) lin-39, a homeobox gene required in QR and its descendents for migration (Wang B. et al, 1993; Clark S. et al, 1993); 2) mig-13, a novel transmembrane protein required outside of QR and its descendents for migration (Sym M et al, 1999); and 3) egl-20, a Wnt homolog expressed in cells in the tail region (Whangbo J.. and Kenyon C, 1999). Two mutant screens (Mary Sym and Queelim Ch'ng) were conducted to identify additional genes that regulate the migration of QR and its descendents. From these screens, mutations in two new genes were identified that cause certain cells in the QR lineage to stop migrating prematurely. These two genes seem likely to be involved in guidance rather than in providing cells with the ability to migrate because, in these mutants, other cells sometimes migrate in the wrong direction. Current efforts are directed toward cloning these two genes and determining how these genes regulate the migration of QR and its descendents together with genes previously known to regulate these migrations. References: Clark SG, Chisholm AD, and Horvitz HR. 1993. Control of Cell Fates in the Central Body Region of C. elegans by the Homeobox Gene lin-39. Cell 74: 43-55. Sym M, Robinson N, Kenyon C. 1999. mig-13 Positions Migrating Cells Along Anteroposterior Body Axis of C. elegans. Cell 98: 26-36. Wang BB, Muller-Immergluck MM, Austin, J, Robinson, NT, Chisholm, A, Kenyon, C. A Homeotic Gene Cluster Patterns the Anteroposterior Body Axis of C.. elegans. Cell 74: 29-42. Whangbo J, Kenyon, C. A Wnt Signaling System that Specifies Two Patterns of Cell Migration in C. elegans. Molecular Cell 4: 851-858.

WB Derry et al. (1999) International C. elegans Meeting "Analysis of the C. elegans homolog of the FHIT tumor suppressor gene"

WB Derry, S van Kreeveld, JH Rothman

[International C. elegans Meeting, 1999]

Alterations in the FHIT gene occur frequently in the development of several human cancers (1). The Fhit protein is a diadenosine P 1 , P 3 -triphosphate hydrolase and is a member of the histidine triad superfamily of nucleotide binding proteins (2). The cellular mechanism of Fhit activity and the relationship between Fhit signaling and tumorigenesis are presently unknown. The C. elegans and Drosophila FHIT genes encode a fusion protein in which the Fhit domain is fused with a novel domain showing homology to bacterial and plant nitrilases, and are referred to as NitFhit (3). We are interested in understanding the role of NitFhit in development and programmed cell death. RNAi of C. elegans NitFhit causes an embryonic arrest phenotype, suggesting an essential role for this gene in development. We are currently analyzing the loss-of-function phenotype and the effect of ectopic NitFhit expression on viability and programmed cell death in the worm. (1) Huebner, K., Garrison, P.N., Barnes, L.D. & Croce, C.M. (1998). Ann. Rev. Genet ., 32 : 7-31. (2) Barnes, L.D., Garrison, P.N., Siprashvili, Z., Guranowski, A, Robinson, A.K., Ingram, S.W., Croce, C.M., Ohta, M. & Huebner, K. (1996). Biochemistry , 35 : 11529-11535. (3) Pekarsky, Y., Campiglio, M., Siprashvili, Z, Druck, T., Sedkov, Y, Tillib, S., Draganescu, A., Wermuth, P., Rothman, J.H., Huebner, K., Buchberg, A.M., Mazo, A., Brenner, C. & Croce, C.M. (1998). Proc. Natl. Acad. Sci. USA , 95 : 8744-8749.

Kenyon CJ et al. (2000) West Coast Worm Meeting "How are anterior cell migrations guided by mig-13?"

Kenyon CJ, Ch'ng Q-L

[West Coast Worm Meeting, 2000]

mig-13 was previously identified as a guidance factor specifically required for the anterior migrations of the QR descendants. mig-13 acts by promoting cell migrations in the anterior direction (Sym et. al., 1999). We are taking several approaches to understand further how the anterior migrations of the QR descendants are guided by mig-13. mig-13 is predicted to encode a novel transmembrane protein containing a CUB domain and LDL-receptor repeat in the extracellular region as well as a proline-rich domain in the intracellular region (Sym et. al., 1999). Our structure/function studies suggest that the extracellular domain of MIG-13 alone can confer partial function in guiding the QR descendants to the anterior. A rescuing mig-13::GFP fusion was previously found to be expressed in the anterior and mid-body ventral cord motor neurons, which cross the migratory track of the QR descendants. Consistent with this expression pattern, mosaic analysis revealed that mig-13 acts non-autonomously to direct the migrations of the QR lineage (Sym et. al., 1999). To determine where mig-13 expression is sufficient to guide the migrating cells, we are expressing mig-13 in broad sets of tissues, as well as in specific subsets of cells that express mig-13::GFP. We also intend to refine previous mosaic analysis to pinpoint the cells in which mig-13 acts. Reference Sym M, Robinson N and Kenyon C. Cell, 1999 Jul 9, 98(1):25-36.

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.

Yung S Lie et al. (2003) International Worm Meeting "Identification of proteins interacting with MIG-13 in Q cell migration"

Jean-Francois Rual, Marc Vidal, Cynthia Kenyon, John R Yates, Scott Anderson, Mary E Hatten, Yung S Lie

[International Worm Meeting, 2003]

We are studying cell migration in the context of anteroposterior migrations of the Q neuroblasts and their descendants. QR and QL are born in right-left symmetrical positions in the animal. QR and its descendants migrate towards the anterior of the body, while QL and its descendants migrate towards the posterior. Proper positioning of QR and its descendants in the anterior requires mig-13. mig-13 encodes a novel transmembrane protein (Sym et al., 1999). We currently know very little about the mechanism by which MIG-13 functions. In order to learn more about how migrating cells determine a final stopping point, we are performing biochemical experiments to isolate proteins that bind to MIG-13. Immunoprecipitations were performed using extracts from worms expressing a rescuing MIG-13-GFP fusion, or as a control, from worms expressing GFP alone. A commercial anti-GFP antibody was used to isolate the fusion protein (or GFP alone in the control) and any associated proteins.Immunoprecipitated samples were analyzed by mass spectrometry to identify proteins present in each sample. We will present data from this mass spectrometry analysis. RNAi was used to disrupt the gene function of candidates identified by mass spectrometry. One such candidate has a QR descendant cell migration phenotype when disrupted by RNAi. We are investigating the interaction of this candidate with MIG-13 by yeast two hybrid and by other biochemical methods. In addition, we plan to complete genetic analysis to determine the functional significance of these protein-protein interactions. We are also currently characterizing a mutant, mu335, which has a phenotype similar to mig-13. QR descendant cell migration is defective in mu335 animals. We have mapped mu335 to the center of LGIII. To identify the gene disrupted by mu335, we are in the process of injecting cosmids for rescue of the migration phenotype. Sym, M., Robinson, N., and Kenyon, C. (1999). MIG-13 positions migrating cells along the anteroposterior body axis of C. elegans. Cell 98, 25-36.

Judkins, Joshua C. et al. (2011) International Worm Meeting "Second generation synthesis and bioactivity of novel endogenous ligands of the nuclear hormone receptor DAF-12."

Judkins, Joshua C., Mahanti, Parag, Bose, Neelanjan, Schroeder, Frank, Hoffman, Jacob

[International Worm Meeting, 2011]

Nuclear hormone receptors (NHR's) are critical components in metazoan signaling cascades, and it is estimated that 284 NHRs exist in C. elegans.1 Although the function of most NHR's may be regulated by small molecule ligands, endogenous ligands for only one C. elegans NHR have been proposed. The two bile-acid-like steroids, D4- and D7-dafachronic acids were predicted as endogenous transactivators of DAF-122, an NHR with high homology to vertebrate Vitamin D and LXR receptors.3 Recent studies by the Schroeder group have revealed the presence of additional ligands of DAF-12. The structural diversity of the newly revealed dafachronic acids highlights the importance of a high yielding and flexible approach to chemically synthesize DAF-12 ligands for biological investigation. Here we present a novel second generation synthesis that is shorter, more efficient, and more versatile than those previously published.4-8 In addition, our synthesis converges on an aldehyde intermediate that allows divergent access to both previously described and novel dafachronic acids. Our synthetic approach provides D4- and D7-dafachronic acids in 6 and 9 steps, respectively. Key features of this synthesis include multiple transformations in a one-pot reaction and a late-stage chiral hydrogenation. The variety of structures attainable through this synthetic method has allowed us to investigate the role of these and related molecules in C. elegans signaling pathways and to examine structure activity relationships (SAR's) of several dafachronic acid variants. We present bioactivity data for several natural and non-natural dafachronic acids. In addition, we compared SAR's obtained from in-vitro experiments with the results from in-vivo studies. [1] Robinson-Techavi et al.; J. Mol. Evol. 2005, 60, 577. [2] Motola et al.; Cell. 2006, 124, 1209. [3] Antebi et al.; Genes & Dev., 2000, 14, 1512. [4] Giroux et al.; Org. Lett., 2008, 16, 3643. [5] Giroux et al.; Org. Lett., 2008, 5, 801. [6] Giroux et al.; JACS, 2007, 129, 9866. [7] Martin et al.; Org. Biomol. Chem., 2010, 8, 739. [8] Gioiello et al.; Tetrahedron, 2011, 67, 1924.

QueeLim Ch'ng et al. (2001) International C. elegans Meeting "How does mig-13 guide anterior migrations?"

Cynthia Kenyon, QueeLim Ch'ng

[International C. elegans Meeting, 2001]

mig-13 is a guidance factor that promotes cell migrations in the anterior direction (Sym et. al., 1999). Previous work demonstrated that mig-13 is required for the anterior migrations of the QR descendants and the BDU neurons (Sym et. al., 1999). Consistent with the role of mig-13 in anterior migrations, we have also found that mig-13 also directs the anterior migration of the distal tip cell (DTC) in the posterior gonad arm during late L3. We are taking several approaches to understand how mig-13 can guide many anterior migrations. mig-13 encodes a novel transmembrane protein containing putative protein-protein interaction domains: a CUB domain and a LDL-receptor repeat in the extracellular region as well as a proline-rich domain in the intracellular region (Sym et. al., 1999). We have examined the function of these domains in MIG-13 by deleting them and assaying the in vivo activity of the resulting MIG-13 construct. Our data suggests that a MIG-13 construct lacking the intracellular domain can confer partial function in directing the QR descendants to the anterior. Previous mosaic analysis revealed that mig-13 acts non-autonomously to direct the migrations of the QR lineage (Sym et. al., 1999). To determine where mig-13 expression is sufficient to guide the migrating cells, we have expressed mig-13 in different sets of tissues, as well as in specific subsets of cells. Expression of mig-13 in all neurons but not any of the other tissues we have tested rescues the QR descendant and DTC migrations in mig-13 mutant animals. This suggests that mig-13 might function in neurons. To pinpoint the cells in which mig-13 acts, we are also refining previous mosaic analysis. For the guidance of the QR descendants, we have narrowed the focus of mig-13 activity to the AB.pr (and possibly the AB.pra) lineage. We have also isolated several mosaic animals that have wild-type QR descendant migrations but mutant DTC migration in the posterior gonad arm, raising the possibility that the migrations of the QR descendants and the posterior DTC are guided by different cells. Reference: Sym M, Robinson N and Kenyon C. Cell, 1999 Jul 9, 98(1):25-36.