Lamkin, E.R. et al. (2017) International Worm Meeting "Morphogenesis of a neuron-glia attachment that shapes dendrite extension."

Lamkin, E.R., McLachlan, I.G., Heiman, M.G.

[International Worm Meeting, 2017]

The function of the nervous system depends on precise connections among neighboring cells, including specialized contacts between neurons and glia. In vivo studies of neuron-glia contacts have been hindered by the challenge of visualizing individual contacts amidst the vast number of cells in the vertebrate nervous system. To study the development of neuron-glia contacts at the single-cell level, we have turned to C. elegans. Two classes of neuron-glia interactions have been studied in C. elegans: (1) glial wrapping of axons in the nerve ring and (2) epithelial-like interactions between sensory dendrite endings and their ensheathing glia. We have focused on a third class of neuron-glia interactions reminiscent of contacts between Bergmann glia and Purkinje cell dendrites in the mammalian cerebellum. The most striking example within this class is the interaction between the BAG sensory neuron and its neighboring glial cell, in which the BAG dendrite ending precisely wraps a protrusion from the glial cell. This interaction was first seen by electron microscopy over four decades ago but has not been further studied. We used super-resolution optical microscopy to visualize the BAG-glia contact in living animals. We also performed forward genetic screens to identify factors required for BAG dendrite development. We showed that the nascent dendrite ending attaches to the glial cell and is pulled to its final length during embryo elongation. Our genetic screens revealed that this attachment requires the cytoskeletal adaptor protein GRDN-1/Girdin, which acts non-cell autonomously in the glial cell to promote dendrite extension in the neuron, and the adhesion molecule SAX-7/L1CAM, which is localized via GRDN-1 to the glial ending where the attachment forms. Based on the partial penetrance of these mutants, we reasoned that additional factors may contribute to the specification and morphogenesis of the BAG-glia contact. Using unbiased and candidate screens for modifiers of the sax-7 dendrite defect we identified roles for the adaptor protein AFD-1/afadin, which is known to localize to cell junctions, the membrane-associated guanylate kinase MAGI-1, and the fibroblast growth factor receptor EGL-15. Our working model is that these molecules form an adhesion complex that specifies the site of the neuron-glia contact. All the adhesion factors we have identified are conserved in mammals, suggesting that mechanisms of neuron-glia contact formation may be shared across systems. We anticipate that, in addition to adhesion factors, other molecules facilitate communication between the neuron and the glial cell to coordinate cell shape changes and generate the precisely interlocking morphologies of the mature contact. The BAG-glia contact thus provides an innovative model for studying the development of a special class of neuron-glia contacts at single-cell resolution.

Chapman, Hannah et al. (2021) International Worm Meeting "Does programmed organismal death promote fitness at the C. elegans colony level?"

Gems, David, Chapman, Hannah, Galimov, Evgeniy R.

[International Worm Meeting, 2021]

Is aging a form of programmed death which, like programmed cell death, promotes fitness? According to evolutionary theory, the answer is no, at least for species that reproduce as outbred dispersed populations. However, it is theoretically possible for adaptive death to evolve in species with clonal, colonial modes of existence, including bacteria, budding yeast and, potentially, C. elegans (1-3). Adaptive benefits of programmed organismal death in post-reproductive adults include increasing food availability for kin (consumer sacrifice) and consumption of dead adults by kin (biomass sacrifice)(1-3). Occurrence of adaptive death in C. elegans could partially explain the presence of so many genes where loss of function increases lifespan (1). We recently created an in silico model of a C. elegans colony on a virtual food patch to probe the likelihood that earlier adult death could increase fitness at the colony level. For the latter, dauer yield per colony was used as a metric. Behavior of the model supported the hypothesis that traits that reduce individual worm fitness can increase colony fitness (4). Increased colony fitness was sometimes observed not only when adult lifespan was shorter but also when brood size was reduced. Notably, reduction of brood size could increase the efficiency with which food was converted into dauer yield. Our findings supported the hypothesis that colony fitness is a function of population structure (4). To test this we have been altering C. elegans colony structure by varying founder animal number, and measuring colony fitness using a dauer yield assay. Results will be presented at the meeting. This work draws attention to the possibility that many characteristics of individual nematodes may promote fitness at the colony level rather than the individual nematode level. (1) J.N. Lohr, E.R. Galimov, D. Gems. Ageing Res. Rev. (2019) 50: 58. (2) E.R. Galimov, D. Gems, Biochem. (Moscow) (2019) 84: 1433. (3) E.R. Galimov, D. Gems, Phil. Trans. R. Soc. London (2021) 376: 20190730. (4) E.R. Galimov, D. Gems, Aging Cell (2020) 19 e13141.

Daniel Omura et al. (2003) International Worm Meeting "Mutations that Cause Food-Deprived Behavior in Well-Fed Animals"

Daniel Omura, Bob Horvitz

[International Worm Meeting, 2003]

Acute food deprivation causes N2 animals to modulate their locomotion differently than well-fed animals upon entering a bacterial lawn1. Acutely food-deprived animals exhibit a serotonin-dependent 'enhanced slowing response' upon entering a bacterial lawn while well-fed animals exhibit a dopamine-dependent 'basal slowing response' upon entering a bacterial lawn. Acute food deprivation has almost no effect on N2 locomotion in the absence of food. We are interested in identifying genes that establish and respond to a food-deprived state. Beth Sawin isolated mutants that do not exhibit food-deprived behavior after an acute food-deprivation event1. Conversely, we sought mutants mutants that constitutively exhibit food-deprived behavior in the absence of acute food-deprivation. To do so we screened for mutants that may inappropriately exhibit a constitutive enhanced slowing response in the absence of acute food deprivation. Our starting strain included a null mutation, n3314, in the mod-5 gene which encodes a serotonin reuptake transporter. This mutation causes a hyper-enhanced slowing response in which mod-5 animals become temporarily paralyzed upon entering a bacterial lawn specifically after acute food-deprivation1,2. We isolated a number of mutants that exhibit a food-dependent paralysis in the absence of acute food deprivation. We then tested for serotonin dependence of this behavior by assaying recovery of locomotion after preincubation with the serotonin receptor antagonist methiothepin. Methiothepin antagonizes the enhanced slowing response, at least in part, by inhibiting the the serotonin-gated chloride channel MOD-13. We screened 5250 EMS mutagenized and 20375 Mos1 transposon mutagenized haploid genomes and isolated 12 mutants, six from each screen. These mutants exhibit a food-dependent paralysis that is antagonized by methiothepin. Most of the mutants grow at a normal rate. Some are slightly Egl. By analyzing genes that underlie the enhanced slowing response, we hope to understand more about how acute food-deprivation modulates C. elegans behavior. 1Sawin, E.R., Ranganathan, R. & Horvitz, H.R., Neuron (2000); 2Ranganathan, R., Sawin, E.R., Trent, C. & Horvitz, H.R., J Neurosci 2001; 3Ranganathan, R., Cannon, S. C. & Horvitz, H. R., Nature (2000).

Jafari, G. et al. (2011) International Worm Meeting "MOD-5/SERT Function in 5-HT-absorbing Neurons Controls Behavioral Response to Food Deprivation."

Xie, Y., Kullyev, A., Liang, B., Sze, J., Jafari, G.

[International Worm Meeting, 2011]

In wild-type hermaphrodites, antibody raised against serotonin stains primarily four classes of neurons in the head region, NSMs, ADFs, RIH and AIMs, and a pair of HSN egg-laying neurons. We found that serotonin is produced only in NSMs, ADFs and HSNs, whereas RIH and AIMs use MOD-5/SERT to absorb serotonin from the extracellular space but cannot synthesize it. To distinguish between these two types of serotonergic neurons, we term NSMs, ADFs and HSNs as "5-HT-producing neurons", and RIH and AIMs as "5-HT-absorbing neurons". By expressing tph-1 specifically in ADFs, or in NSMs in tph-1 mutant background, we found that RIH and AIMs can absorb serotonin released from any of the 5-HT-producing neurons. RIH and AIMs are not connected to the 5-HT-producing neurons (White et al., 1986), suggesting that they absorb 5-HT from extra-synaptic space. Indeed, mutations in either dense-core vesicle release or synaptic vesicle release significantly reduced 5-HT in RIH and AIMs, and mutations affecting both forms of the release produced an additive effect. mod-5 mutants was initially identified in the Horvitz lab based on enhanced slowing locomotion following brief food deprivation (Sawin et al., 2000; Ranganathan et al., 2001). We found that transgenic expression of MOD-5/SERT in AIMs and several non-serotonergic neurons fully corrected the mod-5 mutant phenotype, whereas expression of MOD-5/SERT in NSMs and ADFs had a little effect. Experiments are under way to determine whether these 5-HT-absorbing neurons use serotonin as a "borrowed transmitter" to regulates their own targets, or they subserve to fine-tune the levels of extra-synaptic 5-HT, thereby modulating the targets remote from the serotonin release sites. References Ranganathan, R., Sawin, E.R., Trent, C., and Horvitz, H.R. (2001). Mutations in the Caenorhabditis elegans serotonin reuptake transporter MOD-5 reveal serotonin-dependent and -independent activities of fluoxetine. J Neurosci 21, 5871-5884. Sawin, E.R., Ranganathan, R., and Horvitz, H.R. (2000). C. elegans locomotory rate is modulated by the environment through a dopaminergic pathway and by experience through a serotonergic pathway. Neuron 26, 619-631. White, J.G., Southgate, E., Thomson, J.N., and Brenner, S. (1986). The structure of the nervous system of the nematode Caenorhabditis elegans. Philos Trans R Soc Lond B Biol Sci B 314, 1-340.

Jennifer J Hofmann et al. (2002) European Worm Meeting "Identification of a new mutant defective for DNA damage responses in the C. elegans germ line."

Jennifer J Hofmann, Michael O Hengartner, E Randal Hofmann

[European Worm Meeting, 2002]

The inability to efficiently repair or remove cells with damaged genomes has been linked to several human cancers. DNA damage induces multiple biological responses that insure the integrity of the genome. Checkpoint genes that sense the damaged DNA and transduce signals to the cell cycle, repair and apoptotic machinery mediate these responses. In C. elegans, DNA damage induces cell cycle arrest and apoptosis in the mitotic and meoitic germ cells, respectively. The core apoptotic machinery is required for proper activation of DNA damage induced apoptosis. Furthermore, mutations in several genes have identified a conserved checkpoint pathway that mediates germ cell cycle arrest and apoptosis in response to genotoxic stress1. These include members of the yeast rad' family, hus-1 (E.R. Hofmann and M.O. Hengartner, unpublished) and mrt-2, as well as rad-5, the S. cerevisiea tel2+ homologue2,3. In order to identify new genes involved in DNA damage responses in C. elegans, we employed a genetic screen for mutants defective in ionizing radiation induced cell cycle arrest. In an ongoing screen, we have identified one mutant, op259, which shows multiple defects for DNA damage responses. In addition to a cell cycle arrest defect, op259 is also defective for DNA damage induced apoptosis. Furthermore, op259 has high embryonic lethality, is sterile at 25C and has a weak Him phenotype. op259 is also slow growing and has abnormally small germ nuclei. Mapping experiments have linked op259 to chromosome I. Thus, C. elegans is an excellent genetic tool for identifying new genes involved in DNA damage responses.

Robert Shivers et al. (2007) International Worm Meeting "Isolation and characterization of C. elegans mutants deficient in PMK-1-dependent immunity."

Tristan Kooistra, Robert Shivers, Dennis H. Kim, Bethany Redding

[International Worm Meeting, 2007]

We have previously described a forward genetic approach for the isolation of C. elegans mutants with enhanced susceptibility to pathogens (Esp mutants), which led to the identification of a requirement for a PMK-1 p38 MAPK pathway in pathogen resistance [1]. More recently, we have reported genome-wide microarray analysis of candidate immune effector genes regulated by the PMK-1 pathway and induced by pathogen infection [2]. Based on these data, we generated a strain carrying the integrated transgene agIs219 that expresses GFP under the control of the promoter for the T24B8.5 gene that is regulated by PMK-1 and induced by pathogen infection. We have used this GFP reporter gene in a forward genetic strategy aided by the COPAS fluorescence-activated worm sorter (Union Biometrica) to isolate C. elegans Esp mutants with diminished PMK-1 activation. Thus far, we have screen ~140,000 haploid genomes and have identified 65 mutant isolates. These isolates comprise at least 6 complementation groups, which represent multiple alleles of known PMK-1 pathway components as well as mutants that do not correspond to previously known pathway components. We anticipate that our genetic strategy will identify upstream inputs into the PMK-1 pathway that may function in pathogen recognition and provide insights into the mechanism of action of the Toll-Interleukin-1 Receptor adaptor protein, TIR-1 [3, 4]. 1.Kim, D.H., et al. Science, 2002. 297(5581): p. 623-6. 2.Troemel, E.R., et al. PLoS Genet, 2006. 2(11): p. e183. 3.Couillault, C., et al. Nat Immunol, 2004. 5(5): p. 488-94. 4.Liberati, N.T., et al. Proc Natl Acad Sci U S A, 2004. 101(17): p. 6593-8.

Daniel L Chase et al. (2003) International Worm Meeting "Dopamine-resistant mutants of C. elegans identify components of Go and Gq signaling pathways"

Daniel L Chase, Judy S Pepper, Michael R Koelle

[International Worm Meeting, 2003]

Dopamine, a major neurotransmitter in the mammalian central nervous system, acts through two classes of G protein-coupled receptors known as D1-like and D2-like receptors. These two classes of receptor signal to antagonistically regulate neural activity. Despite intensive research, the signaling mechanisms responsible for the antagonistic effects of dopamine in the brain remain debated. We describe the first genetic screen designed to identify proteins required for dopamine signaling. C. elegans uses endogenous dopamine to inhibit locomotion behavior1, and treatment with excess exogenous dopamine paralyzes worms2. We isolated mutants unresponsive to exogenous dopamine and found that they are also defective for endogenous dopamine signaling. Eleven mutations identified five different genes. Four of these genes encode components of the Go and Gq signaling pathways, including the G protein Go itself and subunits of the RGS complex that inhibits Gq signaling. The Go and Gq signaling pathways act antagonistically in C. elegans to control behavior and their components are conserved in humans. Mutations in these signaling components that decrease Go signaling or increase Gq signaling cause dopamine resistance, while mutations that have opposite effects on these two signaling pathways cause dopamine hypersensitivity. Thus the physiological effects of dopamine are mediated by Go in C. elegans and are antagonized by Gq signaling. Such a dopamine signaling mechanism in mammals could explain many of the opposing effects of D1-like and D2-like receptor activation. Our screen also identified one gene that appears to encode a new Go/Gq signaling component. 1. Sawin, E.R., Ranganathan, R., and Horvitz, H.R. (2000). Neuron 26, 619-631; 2. Schafer, W.R., and Kenyon, C.J. (1995). Nature 375, 73-78.

M Bickle et al. (1999) International C. elegans Meeting "Investigating the sex-specific expression and function of the putative chemosensor srd-1."

M Bickle, D Hughes, J Hodgkin

[International C. elegans Meeting, 1999]

We are analysing the function and expression of the putative chemosensor encoded by srd-1 . srd-1 was identified as a predicted seven transmembrane receptor by Troemel et al in 1995 1 . A fusion of 3750 bp of promoter region of srd-1 to GFP was expressed in the ASI amphid neurons in hermaphrodites whereas in males it was expressed in the ASI, ADF and what appeared to be the R8/9 ray neurons of the tail. We have identified five putative TRA-1A binding sites in the promoter of srd-1 at approximately -810, -910, -2020, -2600 and -3480. TRA-1A is the zinc-finger protein at the end of the sex-determination pathway which is believed to control sex-specific transcription 2 . We mutated these binding sites in a full-length srd-1::GFP fusion kindly provided by Cori Bargmann. Mutation of the TRA-1A binding site at -2600 resulted in the expression of GFP in the ASI and ADF amphid neurons in hermaphrodites, suggesting that TRA-1A might bind directly to the promoter of srd-1 . We are testing this hypothesis by gel-shift assays. We are also investigating the function of srd-1 . We have isolated in collaboration with the Sanger Center a deletion at the srd-1 locus. The deletion covers -583 to +1017 eliminating the first two exons and parts of the third exon thus representing a likely null mutant. The mutant srd-1 is healthy and shows no morphological abnormalities in males or hermaphrodites. It forms dauers and stays on or near the bacterial lawn suggesting it is not impaired in any gross way for chemotaxis and males can mate. We are performing quantitative dauer formation, chemotaxis and mating assays. srd-2 is a close homolog of srd-1 with 48% identity. We will perform RNAi with srd-2 in a srd-1 mutant and test for behavioral alteration. 1.Troemel, E.R., Chou, J.H., Dwyer, N.D., Colbert, H.A. & Bargmann, C.I. Divergent seven transmembrane receptors are candidate chemosensory receptors in C. elegans. Cell 83, 207-218 (1995). 2.. Hodgkin, J. A genetic analysis of the sex-determining gene, tra-1, in the nematode Caenorhabditis elegans. Genes Dev 1, 731-745 (1987).

Suzuki, Michiyo et al. (2013) International Worm Meeting "Quantitative analysis of IR-induced effects on locomotion in Caenorhabditis elegans."

Hattori, Yuya, Sakashita, Tetsuya, Tsuji, Toshio, Suzuki, Michiyo, Kobayashi, Yasuhiko

[International Worm Meeting, 2013]

Background and purpose: C. elegans is a good in vivo model system to examine radiobiological effects. We have previously examined the effects of ionizing radiation (IR) on locomotion in C. elegans using 'body bends' (the number of bends counted in the anterior body region at 20-s intervals) [1] and reported an IR-induced reduction in motility [2],[3]. However, the degree of motility in each region of the body has not been established. In the present study, we employed a video-based analysis and investigated the IR-induced effects on locomotion in more detail. Irradiation and video-based analysis: Young adult wild-type C. elegans were placed on a NGM plate with a bacterial lawn (food) and irradiated with graded single doses (0-1.5 kGy) of 60Co g-rays. Immediately after irradiation, animals were transferred to a NGM plate, either with or without food. The movements of five or more animals placed on each plate were video-recorded. The video images were analyzed off-line based on a previously published method [4]. Briefly, after binarization and denoising, the body line was skeletonized and evenly divided into 12 segments; X- and Y-coordinates of each point on the body were subsequently acquired. To evaluate the motility of each point on the body, the moving distance of each of 13 points over a 5 s period was calculated using the X- and Y-coordinates. In addition, we introduced a novel standard, namely the straight distance from head to tail, to evaluate the body form. Results: Under the -food condition, the moving distance of irradiated animals was reduced in a dose-dependent manner at each point on the body, and there was no difference between the effects on each region of the body. The dose-dependent reduction in locomotion was also observed in animals under the +food condition. Furthermore, we evaluated body form and found that IR-induced quantitative changes in body form. References: [1] Sawin, E.R., et al. (2000) Neuron 26, [2] Sakashita, T., et al. (2008) J. Radiat. Res. 49, [3] Suzuki, M., et al. (2009) J. Radiat. Res. 50, [4] Hattori, T., et al. (2012) Neural Comput. 24.

Tanja Starc et al. (2007) International Worm Meeting "The glutamate gated chloride channel subunit GLC-2 requires AVR-15 and presynaptic glutamate release for proper synaptic localization."

Joseph Dent, Myriam Moujahidine, Cyrus Salek, Tanja Starc

[International Worm Meeting, 2007]

The mechanisms that allow cys-loop ligand-gated anion channels (e.g. glycine and GABA receptors in vertebrates, as well as glutamate, histidine, acetylcholine and serotonin receptors in invertebrates) to localize to synapses is poorly understood. To develop a system in which we can address this question genetically, we generated a full-length genomic clone of the gene encoding the glutamate gated chloride channel (GluCl) subunit GLC-2 with YFP inserted into the intracellular loop. GLC-2 has been reported to be co-expressed in the pharyngeal muscle (Laughton et al.,1997) with another GluCl subunit, AVR-15, and can form a heteromeric channel with AVR-15 in Xenopus oocytes. AVR-15 is necessary postsynaptically for neurotransmission by the glutamatergic pharyngeal motor neuron M3 (Dent et al., 1997). When expressed in Xenopus oocytes, our GLC-2::YFP fusion protein forms homomeric and heteromeric (with AVR-15) channels with only modest changes in glutamate sensitivity and kinetics. In transgenic animals, the GLC-2::YFP construct expressed in the pm3 and pm4 muscles of the pharynx and in the extrapharyngeal neurons RMED and RMEV, a subset of the cells expressing AVR-15. The YFP is in puncta, some of which are localized near to or at the M3 synapse. In an avr-15 null background there is no M3 neurotransmission and no localization of GLC-2::YFP in the muscle or neurons. To determine whether GLC-2 localization requires presynaptic release of neurotransmitter, we placed GLC-2::YFP in an eat-4 mutant background. EAT-4 is the vesicular glutamate transporter in M3 (Lee et al., 1999). In the eat-4(ky5) background, GLC-2::YFP localization is significantly degraded, though not completely eliminated. Interestingly, in the available glc-2 deletion mutants, M3 neurotransmission appears normal. We are currently trying to determine whether GLC-2 forms a heteromeric channel with AVR-15 in vivo. We are also developing screens to identify other localization mutants as well as testing candidate genes. Laughton D.L., Lunt, G.G. and Wolstenholme, A.J. (1997) J. Exp Biol.200:1509. Dent, J.A., Davis, M.W. and Avery, L. (1997) EMBO J. 16:5867. Lee, R.Y.N., Sawin, E.R. Chalfie, M., Horvitz, H.R. and Avery, L. (1999) J. Neurosci. 19:159.