Nathalie Strutz et al. (2001) International C. elegans Meeting "Identification of functional ion channel domains of C. elegans glutamate receptor subunits"

Nathalie Strutz, David Madsen, Michael Hollmann, Andres V Maricq

[International C. elegans Meeting, 2001]

Glutamate receptors are not only abundant in the nervous system of vertebrates but also of invertebrates. In Caenorhabditis elegans , 10 putative ionotropic glutamate receptor subunits have been identified so far (Brockie et al. 2001, J. Neurosci. 21, 1510). Two of these, GLR-1 and GLR-2, have been expressed in Xenopus oocytes and failed to show ligand-activated currents. Therefore, we set out to investigate if the pore-forming domains of these two subunits are intrinsically functional ion conducting domains. We have previously shown that domain transplantation between different subtypes of glutamate receptors can generate functional chimeras that allow the characterization of those domains. To test for pore function, we transplanted the pore-forming region of GLR-1 or GLR-2 into either rat GluR1, as a representative of the AMPA receptor family, or rat GluR6, as a representative of the KA receptor family. The resulting chimeras GluR1-PGLR1, GluR1-PGLR2, GluR6-PGLR1, and GluR6-PGLR2, as well as the respective wildtypes GluR1, GluR6, GLR-1, and GLR-2 were expressed and characterized in Xenopus oocytes. Surprisingly, GluR1-PGLR1, GluR1-PGLR2, and GluR6-PGLR2 produced ligand-activated currents, despite the GLR-1 and GLR-2 wildtype receptor's apparent lack of ion channel function. The maximal current amplitudes of GluR6-PGLR2 were comparable to wildtype GluR6. GluR1-PGLR1 and GluR1-PGLR2 showed reduced current amplitudes in comparison to wildtype GluR1. The ion channel properties of the chimeras classify GLR-1 and GLR-2 into the group of non-NMDA receptor subunits, consistent with conclusions derived from sequence homology data.

Michael A Beer (2005) International Worm Meeting "Systematic genome-wide identification of transcriptional cis-regulatory logic in C. elegans."

Michael A Beer

[International Worm Meeting, 2005]

We have developed a systematic approach for inferring cis-regulatory logic from whole-genome microarray expression data.[1] This approach identifies local DNA sequence elements and the combinatorial and positional constraints that determine their context-dependent role in transcriptional regulation. We use a Bayesian probabilistic framework that relates general DNA sequence features to mRNA expression patterns. By breaking the expression data into training and test sets of genes, we are able to evaluate the predictive accuracy of our inferred Bayesian network. Applied to S. cerevisiae, our inferred combinatorial regulatory rules correctly predict expression patterns for most of the genes. Applied to microarray data from C. elegans[2], we identify novel regulatory elements and combinatorial rules that control the phased temporal expression of transcription factors, histones, and germline specific genes during embryonic and larval development. While many of the DNA elements we find in S. cerevisiae are known transcription factor binding sites, the vast majority of the DNA elements we find in C. elegans and the inferred regulatory rules are novel, and provide focused mechanistic hypotheses for experimental validation. Successful DNA element detection is a limiting factor in our ability to infer predictive combinatorial rules, and the larger regulatory regions in C. elegans make this more challenging than in yeast. Here we extend our previous algorithm to explicitly use conservation of regulatory regions in C. briggsae to focus the search for DNA elements. In addition, we expand the range of regulatory programs we identify by applying to more diverse microarray datasets.[3] 1. Beer MA and Tavazoie S. Cell 117, 185-198 (2004). 2. Baugh LR, Hill AA, Slonim DK, Brown EL, and Hunter, CP. Development 130, 889-900 (2003); Hill AA, Hunter CP, Tsung BT, Tucker-Kellogg G, and Brown EL. Science 290, 809812 (2000). 3. Baugh LR, Hill AA, Claggett JM, Hill-Harfe K, Wen JC, Slonim DK, Brown EL, and Hunter, CP. Development 132, 1843-1854 (2005); Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, Li H, and Kenyon C. Nature 424 277-283 (2003); Reinke V, Smith HE, Nance J, Wang J, Van Doren C, Begley R, Jones SJ, Davis EB, Scherer S, Ward S, and Kim SK. Mol Cell 6 605-616 (2000).

Puri, Dharmendra et al. (2017) International Worm Meeting "Regulation of neuronal microtubule cytoskeleton."

Jin, Yishi, Puri, Dharmendra, Chisholm, Andrew, Ghosh-Roy, Anindya, Thyagarajan, Pankajam

[International Worm Meeting, 2017]

In the nervous system, directional flow of information depends on the polarized architecture of neuron. Neuron has a unique structure - many dendrites and one axon. Microtubule polymers are the building blocks of the polarized architecture of neuronal cell. Although MT protofilaments give stability in axon, they themselves are dynamic - undergoing polymerization and depolymerization. The dynamics of the microtubule network needs to be regulated in a context-dependent manner during polarization, maintenance or other remodeling processes. Loss of kinesin-13 family microtubule depolymerizing enzyme klp-7 (kinesin-like protein) leads to excess stabilization of microtubules, leading to a multipolar neuronal phenotype in C. elegans mechanosensory (1) and other neurons. We found that this phenotype in klp-7(lf) can be reversed by a microtubule-destabilizing drug Colchicine or in backgrounds lacking either alpha or beta tubulin. We hypothesized that a genetic screen for the suppressors of klp-7(lf) will help us identify regulators of microtubule cytoskeleton. We conducted a genetic screen saturating the mutagenized genome. By combining meiotic recombination mapping and whole genome sequencing, we have identified candidates such as tubulin genes, post-translation modification factors and other novel factors. We mapped one of the suppressors to a gene, muscleblind-1/mbl-1. We found that mbl-1 is necessary and sufficient for touch neuron axon extension in a cell-autonomous manner. Gentle touch response in posterior and anterior side is compromised in mbl-1 mutant. Mbl-1 function is mostly known in muscles as a splicing regulator. Recent study in worm suggested that MBL-1 is required for synapse stabilization in DA9 motor neuron (2). We hypothesize that MBL-1 is regulating splicing of genes required for microtubule stability. References: 1. Ghosh-Roy A, Goncharov A, Jin Y, & Chisholm AD (2012) Kinesin-13 and tubulin posttranslational modifications regulate microtubule growth in axon regeneration. Dev Cell 23(4):716-728. 2. Spilker KA, Wang GJ, Tugizova MS, & Shen K (2012) Caenorhabditis elegans Muscleblind homolog mbl-1 functions in neurons to regulate synapse formation. Neural Dev 7:7.regulation

Andersen*, Kristine et al. (2015) International Worm Meeting "An Investigation into the Affect of Neuronal Activity on Proper Neural Connectivity in C. elegans."

Andersen*, Kristine, Varshney, Aruna, Duong, Alex, Miller, Kristine, Park, Joori, Pyle, Jacqueline, Barsi-Rhyne*, Benjamin, VanHoven, Miri, Vargas, Christopher, Holdrich, Emma, Tsujimoto, Bryan, Tran, Alan

[International Worm Meeting, 2015]

Neuronal activity has been implicated in the establishment and maintenance of appropriate synaptic connections in vertebrate and invertebrate systems. However, the molecular mechanisms by which neuronal activity affects connectivity are poorly understood. To understand this process, we have focused our studies on the PHB phasmid sensory neurons. We have begun to elucidate the pathway by which sodium dodecyl sulfate (SDS) is sensed by the phasmid neurons using a high throughput assay adapted from the method developed by Hilliard and colleagues (Current Biology, 2002). Briefly, SDS is sensed by the PHB neurons, which terminate backward movement via their connections with AVA interneurons. Using this assay, we find that odr-3/Galphaolf and tax-2/CNG-channel beta subunit, in addition to previously discovered tax-4/CNG-channel alpha subunit (Hilliard et al., Current Biology, 2002), are required for SDS chemosensation. To determine if defects in sensory signaling affect sensory synapses, we utilized the split-GFP-based trans-synaptic marker NLG-1 GRASP (Neuroligin-1 GFP Reconstitution Across Synaptic Partners) to visualize synapses between PHBs and AVA interneurons in live animals. Interestingly, we find that neuronal activity is required for maintaining these sensory synapses. Time course experiments indicate that odr-3/Galphaolf mutant L1s have normal synapses, but synapses are significantly reduced in later larval stages, although phasmid neuron morphology appears to be unaffected. Cell-specific rescue experiments indicate that odr-3/Galphaolf likely functions in PHB neurons. Subcellular localization of odr-3/Galphaolf shows localization to the PHB cilia, consistent with a role in sensory signaling being required to maintain synaptic integrity. These results indicate that C. elegans may be a powerful model organism for elucidating the molecular mechanisms by which sensory activity affects synaptic connectivity. Our future goal is to further characterize the mechanism by which sensory activity maintains synapses using molecular genetic and physiological approaches. Funded by NIH (1R01NS087544 to MV at SJSU and NL at UCSF, 5T34GM008253 MARC undergraduate fellowship to CV, 2R25GM071381 RISE undergraduate fellowships to CV and JP), HHMI (SCRIBE 52006312 undergraduate fellowship to BB and KM), and NSF (RUMBA REU 1004350 fellowship to KA and EH).

Vidal-Gadea, Andres G et al. (2011) International Worm Meeting "C. elegans selects distinct crawling and swimming gaits via dopamine and serotonin."

Elbel, Erin, Axelrod, Abram, Erbguth, Karen, Topper, Stephen, Brauner, Martin, Vidal-Gadea, Andres G, Crisp, Ashley, Pierce-Shimomura, Jonathan, Young, Layla, Siegel, Dionicio, Maples, Thomas, Kressin, Leah, Gottschalk, Alexander

[International Worm Meeting, 2011]

The ability to select and switch between alternate locomotory gaits is shared among many animals that are required to transition between different environments. Failure to achieve motor transitions is often devastating to the organism as exemplified by patients with advanced Parkinson's disease. The nematode C. elegans is well adapted for locomotion on land and in water and is likely required to transition between these sub-niches in nature. Despite recent advances, it remains controversial if swimming and crawling are distinct gaits in C. elegans, and if so, how transitions between them are accomplished (1-4). Answering these questions could enable the use of this powerful model system to study the mechanisms underlying locomotory transitions. We used a multifaceted approach spanning in depth behavioral assays, mechanic and optogenetic stimulation, laser ablations, mutant analysis, and in vivo photolysis of caged amines to test whether the worm uses distinct gaits and to determine how they transition between them. We show through a series of behavioral assays that in C. elegans crawling and swimming are distinct gaits. Dopamine released by ADE and PDE neurons is necessary and sufficient to initiate and maintain crawling by a pathway activating D1-like receptors. Conversely, serotonin is necessary and sufficient to initiate and maintain transition from crawling to swimming and to inhibit a set of crawl-specific behaviors. We found these transitions to be modulated by the balance between dopamine and serotonin. These amines have been found to play crucial roles in transition between alternate locomotory forms in diverse species stressing the importance locomotor transitions have for survival. Further study of locomotory switching in C. elegans and its dependence on dopamine may provide insight into comparable motor deficits observed in Parkinson's disease. 1) Boyle H et al. N. Front. Behav. Neurosci. 2011 5:10 doi: 10.3389/fnbeh.2011.00010. 2) Mesce KA, Pierce-Shimomura JT. Front. Behav. Neurosci. 2010 4:49 doi: 10.3389/fnbeh.2010.0004 3) Vidal-Gadea AG et al. 2009 Neuroscience 366:17 Calabrese R: Faculty of 1000 Biology, 11 November, 2009. Conference 2009 October 16-21. Available at: http://f1000biology.com/ article/id/1182956/evaluation 4) Fang-Yen C et al. 2010 PNAS 107, 20323-20328.

Hsieht J et al. (1996) East Coast Worm Meeting "A Screen For Mutants Which Affect Repeat-Induced Gene Silencing in C. Elegans"

Fire A, Hsieht J

[East Coast Worm Meeting, 1996]

We are studying an apparent silencing phenomenon associated with tandemly arrayed repetitive sequences in C. elegans. Inactivation of repeated genes appears to be a widespread occurrence in a variety of eukaryotes. In Drosophila, genes repeated and/or juxtaposed to heterochromatin are frequently inactivated (1, 2), a phenomenon known as positioneffect variegation. In plants, silencing interactions between duplicated genes, otherwise known as repeatinduced gene silencing, are also commonly observed (3, 4). In a previous worm breeders' gazette (5), our lab reported that certain expression constructs are silenced when introduced into the animal as extrachromosomal tandem arrays of plasmid sequences; silencing is relieved by introduction of the transgene in a nonrepetitive context. We observe silencing in both some and gennline. Germline expression of the gene let858 is particularly sensitive to the effect, but we also observed silencing of certain constructs that are otherwise active in the some (5). In order to examine the mechanisms recognizing and acting on tandemly repeated sequences in C. efegans, we would like to isolate mutants that affect the apparent gene silencing. We are using a plasmid construct (pPD98.41) in which a gfp reporter is driven by a combination of the unc-54 enhancer and myo-2 promoter. In nonrepetitive situations (either F1 assays or lines in which the transgene is present in a nonrepetitive context) we see expression of the construct in both body wall and pharyugeal muscle. From a tandem array of pPD98.41 mixed with the rol6 marker plasmid pRF4, we see only expression in the pharynx. We interpret this as indicating that the ability of the unc-54 enhancer to activate the myo-2 promoter is silenced in repeated sequence contexts. Our general scheme is to mutagenize lines carrying these tandem arrays and then to look in subsequent generations for animals in u hich the transgene is no longer silenced. We are using a nonclonal approach to screen for viable mutations and a clonal approach to recover potentially lethal or sterile mutations. In preliminary screens, we have begun to recover animals that exhibit increased bodywall muscle expression of file transgene. Lines derived from such animals will he characterized to determine whether changes have occurred in the array, in muscle gene regulation, or in the silencing process. In a parallel set of experiments, we have been addressing the question of whether the extrachromosomal nature of arrays is involved in silencing. We are using gamma-irradiation to integrate the arrays described above. In the first integrants obtained, we have not observed any effect of the integration on the ability of the array to be silenced. 1 Spofford, J.B. (1976), Genetics and Biology of Drosophila, Volume lc: 9551019; 2 Henikoff, S., Dorer, D.R. (1994), Cell 77: 9931002; 3 Matzke, M.A., Matzke, A.J.M., and Mittelsten Scheid, O. (1994), Homologous Recombination in Plants 271307; 4 Assaad, F.F., Tucker, K.L., and Signer, E.R. (1993), Plant Molecules Biology 22: 1067lO85; 5 Kelly, B., Xu, S., Fire, A. (1995), Worm Breeders Gazette, 14(1): 6465