Scott Everet Baird et al. (2004) East Coast Worm Meeting "Association of Oscheius species with millipedes in the Wright State University Woods."

Scott Everet Baird, Christine M Spice

[East Coast Worm Meeting, 2004]

Rhabditid nematodes commonly form phoretic or necromenic associations with other soil invertebrates. These associations typically are as dauers although although associations of other larval stages also have been reported. Two hermaphroditic species of Oscheius , O. myriophila and an isolate tenatively identified as O. necromena , have been reported as necromenic associates of millipedes. Both of these species have been found in association with the millipede Pseudopolydesmus serratus in the Biology Preserve on the campus of Wright State University. A third as yet unnamed hermaphroditic species of Oscheius was found in association with a second millipede, Oxidus gracilis . O. myriophila , O. necromena , and O. sp. were found at the same collection site, i.e. they were sympatric. O. myriophila never was found in association with Ox. gracilis despite this millipede being its type host. When first observed, all collected nematodes were L4s, consistant with a previous report of the association of O. myriophila L4s with millipedes. Relationships among these species were investigated through sequence comparisons of 18S rDNA. O. myriophila , O. necromena , and O. sp. clustered together as a monophyletic clade within the insectivora -group of Oscheius . Thus, association with millipedes and their hermaphroditic mode of reproduction may be ancestral characters of this group of species.

Pettitt J et al. (1995) International C. elegans Meeting "pun-1(ct359): A MUTATION AFFECTING PHARYNX ATTACHMENT TO THE BUCCAL HYPODERMIS"

Pettitt J, Edgar LG, Wood WB

[International C. elegans Meeting, 1995]

We have isolated a psoralen-induced mutation at a locus we have termed pun-1 (pharynx unattached). pun-1(ct359) homzygotes arrest as L1 larvae displaying variable defects in body morphology, ranging from a mild dumpy phenotype to animals which appear to have failed to elongate properly and have a severely disrupted hypodermis. ct359 homozygotes, however, also exhibit a failure of the buccal capsule (the cuticle lining the buccal cavity) to attach to the buccal hypodermis. This phenotype is similar to that observed in animals transgenic for a truncated version of cdh-3, a cadherin-related gene (see other abstract by Pettitt et al.). We are now using various markers to more fully characterize the pun-1(ct359) phenotype. In particular we would like to determine which cells are involved in the Pun phenotype and confirm that the phenotype is due to a failure of cell adhesion and not merely the absence of the cells involved. The anterior part of the buccal capsule is secreted by the arcade cells and the posterior part by the pharyngeal epidermal cells (Wright and Thompson, Can. J. Zool. 59: 1952-196). We assume that the arcade cells, and/or hyp1 cells are affected in pun-1 homozygotes, since the unattached pharynx in these animals retains the buccal capsule, suggesting that the pharyngeal epidermal cells are unaffected. pun-1 has been mapped to LGI and we are currently refining its map position.

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.

Yohann Duverger et al. (2007) International Worm Meeting "A semi-automated high-throughput approach to the generation of transposon insertion mutants."

Yohann Duverger, Jonathan Ewbank, Christophe Beclin, Sarah Scaglione, Dominique Brandli, Jerome Belougne

[International Worm Meeting, 2007]

To understand gene function, there is a clear need for the generation of as many stable mutant lines in as possible. Two KO projects are currently underway in North America (http://celeganskoconsortium.omrf.org/) and Japan (http://shigen.lab.nig.ac.jp/c.elegans/index.jsp) and have generated thousands of mutants. To these can be added the null alleles for several hundred genes produced using other PCR-based technologies or generated in classical genetic screens and available from the CGC. An alternative approach to the generation of mutants is via the use of transposons. Recently, a method using the mariner-like element Mos1 from Drosophila has been established (1). It was used in a pilot-scale project to generate random insertions throughout the genome (2). Following on from this manually project, as part of a collaborative effort funded by the European community (Nemagenetag; see abstract by Robert et al.), the French National Genopole network and support from Union Biometrica, we have developed a semi-automated high-throughput method for mutant production and screening. Using the UBI COPAS Biosort and a robotic system for PCR and electrophoretic analysis (using a 400-well gel format), we have developed a capacity to handle several thousand nematode strains in parallel for multiple generations (3) and have already generated more than 45,000 individual strains carrying Mos1 insertions. We will the describe method in detail, report recent improvements that have pushed our production capacity to above 5,000 mutant strains/month and explain how the method can be easily adapted to forward and reverse genetic screens. 1. Bessereau, J. L., Wright, A., Williams, D. C., Schuske, K., Davis, M. W. & Jorgensen, E. M. (2001) Nature 413, 70-4. 2. Granger, L., Martin, E. & Segalat, L. (2004) Nucleic Acids Res 32, e117. 3. Duverger, Y., Belougne, J., Scaglione, S., Brandli, D., Beclin, C. & Ewbank, J. J. (2007) Nucleic Acids Res 35, e11.

Loer CM (1995) International C. elegans Meeting "DOES THE SEROTONIN- AND DOPAMINE-DEFICIENT MUTANT CAT-4 ALSO HAVE A LEAKY CUTICLE?"

Loer CM

[International C. elegans Meeting, 1995]

The mutant cat-4 (e1114)V was first isolated as a dopamine-deficient mutant lacking formaldehyde-induced fluorescence (FIF); it was subsequently found to lack serotonin immunoreactivity (Sulston et al., 1975, J. Comp. Neurol. 163: 215; Desai et al., 1988, Nature 336: 638). Although cat-4 mutant worms were previously described as serotonin hypersensitive, we have found that cat-4 mutants are also hypersensitive to a variety of other unrelated agents. We first noticed that cat-4 mutant worms died much more rapidly than wild type worms during routine "cleaning" of worms with bleach. Whereas wildtype worms squirm in bleach for several seconds before becoming rigid, cat-4 mutants stop moving almost instantly (< 1 sec). In addition, cat-4 mutant carcasses seem to disappear/dissolve during this treatment, unlike those of wild type worms. These observations suggest that mutation of the cat-4 gene affects not only produc-tion of neurotransmitters, but also construction of the cuticle. We confirmed that cat-4 mutants are hypersensitive to serotonin in an egg-laying assay. Although there was no difference in the number of eggs laid when cat-4 mutants were compared with wild type in 10 mM serotonin, cat-4 mutant laid significantly more eggs than wild type in 5 mM or 2 mM serotonin. We also found differences between cat-4 mutants and wild type in acute survival in both SDS and levamisole. This general hypersensitivity suggests that cat-4 mutants have a more permeable cuticle than wild type worms. Among the non-reducible cross-links found in nematode cuticle proteins are those formed by isotrityrosine (reviewed by Cox, 1992, J. Parasitol. 78: 1). The defects in cat-4 mutants suggest that the nervous system and hypodermis share enzymatic pathways for synthesis of biogenic amine neurotransmitters and cuticle cross-linking agents. Although nematode and insect cuticles are very different (collagenous vs. chitinous, respectively), it may be that aromatic amino acid derivatives are used by nematodes similarly to insects (Wright, 1987, Adv. Genet. 24: 127).

Chenggang Lu et al. (2003) International Worm Meeting "C. elegans genes tbb-2 and tba-2 are preferred tubulin isotype substrates of the MEI-1/MEI-2 microtubule severing complex during meiotic spindle formation."

Chenggang Lu, Paul Mains

[International Worm Meeting, 2003]

Female meiotic spindle formation requires two meiosis-specific components, MEI-1 and MEI-2, which are similar to the p60 (severing) and p80 (localization) subunits, respectively, of the sea urchin microtubule-severing complex katanin. In addition to their sequence similarities to katanin, MEI-1 and MEI-2 disassemble interphase microtubules when coexpressed in Hela cells.1 In wild-type embryos, MEI-1 and MEI-2 localize exclusively to the female meiotic spindle. MEI-1 and MEI-2 likely regulate the length of meiotic spindle fibres to maintain the unique morphology of the small anteriorly-located, barrel-shaped meiotic spindle, but MEI-1/MEI-2 must be inactivated prior to mitosis. Here we report analysis of three extragenic suppressors of a mei-1(gf) mutant that results in ectopic microtubule-severing activity during mitosis. These suppressors show semi-dominant suppression of the mei-1(gf) defects, but are phenotypically wild type by themselves. One suppressor is a missense allele of the -tubulin gene, tbb-2 (C36E8.5). The other two are missense alleles of an -tubulin gene, tba-2 (C47B2.3). All three suppressors genetically behave as if they generally inhibit microtubule severing: they suppress the mitotic phenotype resulting from ectopic mei-1 activity while enhancing meiotic defects seen when MEI-1 severing activity is compromised during meiosis. Although functional redundancies were seen with and -tubulins during worm development (our data, Philips et al. and Wright et al.), our experiments revealed tubulin isotype preferences of the MEI-1/MEI-2 severing complex. When MEI-1 and MEI-2 are limiting, tbb-2(RNAi) resulted in meiotic defects while tbb-1(RNAi) did not. Thus, MEI-1/MEI-2 prefers the TBB-2 isotype as a substrate. Similarly, TBA-2 is the preferred -tubulin substrate. Therefore, although TBB-1/TBB-2 and TBA-1/TBA-2 isotype pairs are functional redundant, our results reveal differences in their functions. 1. Srayko, M., Buster, D.W., Bazirgan O.A., McNally, F.J. and Mains, P.E. (2000). Genes Dev. 14, 1072-1084.

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

LS Kaltenbach et al. (1999) International C. elegans Meeting "tlp-1 encodes a variant TATA-binding protein that is essential for embryogenesis"

M Domeier, SE Mango, LS Kaltenbach

[International C. elegans Meeting, 1999]

One component of the basal transcription machinery is a 700kD complex called TFIID. TFIID is composed of TATA-binding protein (TBP; CeTBP in C. elegans 1 ) and several TBP-associated factors. The prevailing view is that this complex is expressed in all cells, at all stages, and that it plays an important role in promoter recognition and recruitment of additional components of the basal machinery to target genes. We have identified a variant of TBP (called tlp-1 for T BP- l ike p rotein) in a yeast two-hybrid screen designed to identify proteins that interact with conserved C-terminal sequences of the pharynx identity factor PHA-4 2 . TBP variants have also been found in flies, humans and rodents 3 . Given the ubiquitous and fundamental role of TBP during transcription, why do animals need a second TBP-like protein? Our data suggest that tlp-1 has an essential function during embryogenesis. First, we examined tlp-1 expression. We defined the tlp-1 gene structure and constructed a tlp-1::GFP chimera. This gene is expressed widely from early embryogenesis until adulthood. By contrast, CeTBP::GFP expression does not initiate until the 3-fold stage. Second, we used RNA interference to determine the loss-of-function phenotypes of tlp-1 and CeTBP . The phenotype of most tlp-1(RNAi) embryos mimics the effects of blocking RNA polymerase II 4 : expression of an early zygotic GFP reporter construct is extinguished, E and MS blastomeres fail to gastrulate normally, and embryos arrest at about the 100-cell stage with little overt differentiation. CeTBP(RNAi) embryos, on the other hand, progress through embryogenesis and arrest as fully differentiated L1 larvae. In summary, we propose that i) PHA-4 activates transcription by targeting the basal transcription machinery via the PHA-4 C-terminus and ii) variant TBP-like proteins play an essential role during embryogenesis to mediate RNA polymerase II-dependent transcription. 1. Lichsteiner and Tjian, 1993. 2. Thanks to Bob Barstead and Phillip James for yeast two-hybrid reagents. 3. Crowley et al., 1993; Hansen et al., 1997; Ohbayashi et al., 1999. 4. Edgar et al, 1994; Powell-Coffman et al., 1996. Thanks to Spencer Wright for technical assistance.

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.