Marsili, J. et al. (2017) International Worm Meeting "The nematode Caenorhabditis elegans displays a chemotaxis behavior to tuberculosis-specific odorants."

Nguyen , Q.H., Voisine, C., McFall , S.M., Marsili, J., Neto, M.F., Aldakeel , S.

[International Worm Meeting, 2017]

The World Health Organization's Millennium Development Goal of halting and reversing the tuberculosis (TB) epidemic by 2015 has been met; on average, incidence of TB has fallen 1.5% per year and is now 18% lower than in 2000. Despite these gains, TB remains one of the leading causes of morbidity and mortality globally-in 2014, 9.6 million people fell ill with TB and 1.5 million died from the disease. Each year, approximately one third of the TB infected population remain undiagnosed, equating to roughly 3 million people. A simple, affordable diagnostic test for pulmonary tuberculosis (TB) is urgently needed to improve detection of active Mycobacterium tuberculosis. Recently, it has been suggested that animal behavior can be used as a biosensor to signal the presence of human disease. For example, the giant African pouched rats can detect tuberculosis by sniffing sputum specimens while honeybees respond to three of the four tuberculosis-specific volatile organic compounds (TB-VOCs) detected in the breath of TB positive patients by proboscis extension. However, both rats and honeybees require animal housing facilities and professional trainers, where few laboratories in low-income, high-burden TB settings possess the needed infrastructure. Rather than using trained animals, we propose exploiting the innate sensing capabilities of the nematode Caenorhabditis elegans, which has demonstrated reproducible, behavioral responses to over 100 VOCs. Our data shows that the innate olfactory behavioral response of C. elegans can be used to detect the TB-specific VOCs methyl p-anisate, methyl nicotinate, methyl phenylacetate and o-phenylanisole, in chemotaxis assays. It is important to note, the breath of a TB patient contains a heterogeneous mixture of TB-VOCs, although the proportionality of this blend remains unknown. To further evaluate the efficacy of C. elegans as a potential TB biosensor, we also investigated C. elegans' behavioral response to combinations of TB-VOCs, both pairwise and collectively. We found that C. elegans detects a mixture of all TB-VOCs, exhibiting a significant avoidance behavior. In addition, dauer larvae, a long-lived stress resistant alternative development state of C. elegans in which the animals can survive for extended periods of time in dry conditions with no food, were also demonstrated to detect the VOCs. Taken together, we propose a potential work-flow for a noninvasive diagnostic test for TB that incorporates collecting VOCs from the breath of the patient, exposing dauer larvae housed in a microfluidic device to the VOCs, and recording their response. These results showcase the potential for C. elegans to be incorporated into a new and affordable diagnostic test device for TB and, perhaps, other disease states, capitalizing upon its innate olfactory responses.

Mark Steciuk et al. (2003) International Worm Meeting "Synaptic Transmission in the Evolution of Behavior."

Alan Chiang, Mark Steciuk, Leon Avery

[International Worm Meeting, 2003]

The pharynx, is divided into three functional compartments: the corpus, the isthmus, and the terminal bulb (TB). In C. elegans the corpus and the TB are connected via gap junctions, resulting in synchronous contractions of these compartments. Feeding behavior is regulated by 20 pharyngeal neurons. In different nematode species, different subsets of synapses function to regulate feeding behavior. In C. elegans, M4 innervates the isthmus and the TB and is necessary for proper isthmus function, but has no detectable TB function. In P. redivivus the TB is capable of pumping independently of the corpus. This behavior is not present in C. elegans and it requires the motor neuron M4. So we propose that different subsets of M4 synapses are acting to regulate feeding behavior in C. elegans and P. redivivus. Our analysis of slo-1 mutants in C. elegans suggests that differential activity of M4TB synapses between the two species is due to differences in synaptic strength. SLO-1 is a Ca-activated K channel. slo-1 mutants have novel M4 dependent EPSPs on EPGs (electrical recordings of the corpus and TB). slo-1 mutants have increased TB pumping in an eat-5 background (loss of eat-5 isolates the TB electrically by inactivating gap junctions between the corpus and TB), and M4 and M5 are the only known neurons to innervate the TB. slo-1 has been shown to increase synaptic transmission at body wall NMJs (Wang et. al. 2000). We interpret our results as activation of the normally silent M4TB synapses in slo-1 mutants. Thus, simply by increasing synaptic transmission, C. elegans M4TB synapses can be made functionally similar to proposed homologous synapses in P. redivivus. This raises the possibility that slo-1 may be important in the functional evolution of M4TB synapses. In addition to activating the M4TB synapse, slo-1 has another interesting phenotype. eat-5 mutants grow slowly on DA837 (hard to eat E. coli). slo-1 suppresses the growth rate defect of eat-5 on DA837, likely due to increased TB pumping. To find other genes that may activate the M4TB synapse we screened for eat-5 suppressors and identified unc-2 and unc-36. UNC-2 and UNC-36 are Ca channel subunits, suggesting a role for voltage-gated Ca channels in the silencing of the M4TB synapse. Since SLO-1 is a Ca-activated K channel, we believe that UNC-2 and UNC-36 may be acting upstream of SLO-1. This result is surprising since mutations in these genes would be expected to decrease synaptic transmission. We hypothesize that other Ca channels are important for synaptic transmission at the M4TB synapse.

Leon Avery et al. (2001) International C. elegans Meeting "M5 controls terminal bulb contraction in Panagrellus redivivus but not in C elegans"

Leon Avery, Mark Steciuk

[International C. elegans Meeting, 2001]

In the C elegans pharynx contractions of the corpus (first bulb) and terminal bulb (TB) are synchronized because excitation begins in the corpus and spreads through gap junctions to the TB. In Panagrellus redivivus the TB pumps three times faster than the corpus, suggesting that the Panagrellus TB must have another source of excitation. Albertson and Thomson's EM reconstructions of the C elegans pharynx showed that a single motor neuron, M5, synapses onto TB muscles. However, M5 has little or no detectable function in C elegans . When we killed M5 in Panagrellus , TB contractions were at the same rate as and synchronized with corpus contractions, as in C elegans . Why might it be advantageous for the Panagrellus TB to pump faster than the corpus? The innexin EAT-5 couples the C elegans corpus and TB. In an eat-5 null mutant the TB is unsynchronized with the corpus and pumps more slowly. eat-5 mutants grow almost normally on E. coli HB101 (easier to eat than most E. coli strains) but can't grow on DA837 (a derivative of OP50). By selecting for suppressors of eat-5 that allowed growth on DA837, we isolated one strain, sef(ad1614) eat-5 , in which corpus and TB are still unsynchronized, but the TB pumps about twice as fast as in the eat-5 single mutant. This result suggests that the rate of TB pumping determines growth on DA837. We hypothesize that the faster TB pumping in Panagrellus may allow it to use low-quality food sources. Each of several different nematode species that we have tested grows faster on HB101 than DA837, but the difference varies widely from species to species. Panagrellus is less affected by the food source than any other species tested. To find other differences between pharyngeal nervous system function in the two species we killed each of the 14 pharyngeal neuron types in each of 5-10 worms of each species, then measured the time required to grow to adulthood on DA837. By this crude test two C elegans pharyngeal neuron types are important: M4, essential for contraction of posterior isthmus muscles, and MC, which controls the rate of pumping. In Panagrellus 2/12 neuron types tested affected growth rate: M2 and M5. In C elegans M2 innervates isthmus muscles but has no detectable effect on pharyngeal function. We have not yet determined the feeding defect in M2-minus Panagrellus . M4 had no detectable effect on Panagrellus growth. (For technical reasons, MC and I2 have not yet been tested.) Thus, in both species only a small subset of pharyngeal neuron types is important for grossly normal feeding, but the important subsets are different. Nematodes have anatomically similar nervous systems despite wide variations in behavior. For instance, the locomotory nervous systems of adult C elegans and Ascaris lumbricoides are cell-for-cell identical, despite the great difference in size and locomotory behavior (Stretton et al). How can the same nervous system produce different behavior? Based on our results we propose the following ROGUT ( R idiculously O versimplified G rand U nified T heory): The 118 neuron types in the adult female nematode represent a menu of possible behaviors. In any given species, only a subset of these types actually functions; which subset determines behavior. While this idea cannot explain all variation in nematode behavior, we believe it may be part of the explanation. The most controversial implication of this hypothesis is that a neuron type may persist through evolution in the absence of selection for its function. It is possible that M5 (for instance) has an important function in C elegans in the wild that we cannot detect in the lab. If so, that function must be different from its function in Panagrellus , since M5 function in Panagrellus is easily detected in the lab. But if M5 has a function in C elegans different from its function in Panagrellus , it is surprising that its function in Panagrellus is predicted by its anatomy in C elegans . In any case, the substantial differences in pharyngeal nervous system function we have discovered are difficult to reconcile with the hypothesis that nematode nervous system constancy is maintained by selection for function. Instead we favor the hypothesis that nematodes have similar nervous systems because of developmental constraints.

Bossinger O et al. (1995) International C. elegans Meeting "EARLY EMBRYONIC INDUCTION AND ENDOCYTOSIS OF THE GUT PRI- MORDIUM CAN BOTH BE INHIBITED BY ACID BISAZO DYES"

Schierenberg E, Bossinger O

[International C. elegans Meeting, 1995]

Acid bisazo dyes bind strongly to the plasma membrane. Ta- king into account their negative charges a mechanism has been postulated by which they bind to positively charged side chains of surface molecules, thus inhibiting e.g. the coupling of ligand to its receptor. We tested the inhibitory effect of Trypan Blue (TB) during two developmental events: first, during the process of gut induction, which occurs between the 4- and 6-cell stage and second during the en- docytotic activity of the gut primordium starting in the late proliferation phase. After application of TB via laser-penetration prior to the 6-cell stage we could in fact inhibit the typical polarisa- tion of the EMS cell through the germline cell P2 with the following results: (1) The EMS cell divides with a transverse instead of an anterior-posterior spindle orientation . (2) Division rhythms of E and MS are the same instead of different. (3) The typical birefringent granules in the gut primordium do not develop. After the 6-cell stage TB does not influence the development of the gut primordium anymore. We have started to investi- gate whether other cellular interactions (e.g. induction of the pharynx) are also affected by TB. During further development gut differentiation in C. elegans goes along with the accumulation of yolk proteins. An exo- / endocytosis mechanism from the non-gut cells into the gut primordium has been hypothesized. We have tested the gut primordium for receptor-mediated endocytosis by using fluo- rescently labelled human transferrin (TF) as a marker. After laser-penetration TF is rapidly internalized from the pe- rivitelline space into the gut primordium. Also here TB is very effective by inhibiting endocytotic activity of the gut primordium in a concentration dependent manner.

Bossinger O et al. (1991) International C. elegans Meeting "FLUORESCENT VITAL DYES AS USEFUL MARKERS IN C. ELEGANS DEVELOPMENT"

Bossinger O, Schierenberg E

[International C. elegans Meeting, 1991]

We have established three different techniques (incorporation with food; microinjection; laser penetration of the eggshell) for introduction of fluorescent dyes into embryos and adults of C. elegans to visualize different developmental processes. Embryos labelled in this way can develop normally, hatch and reproduce. After feeding animals with Neutral Red, a marker for lysosomes, the dye concentrates in the autofluorescent gut granules. The fluorescent dye Lucifer Yellow (LY) can be used as a useful marker for the presence of yolk. If injected into the body cavity of adult hermaphrodites it stains e, Yclusively oocytes in a graded fashion. Only the most mature oocytes are prominently labelled. With the help of LY we can visualize a stage- specific uptake process into the gut primordium. If the eggshell of an early embryo is laser-penetrated in presence of LY in the surrounding medium, the fluorescent dye will fill the perivitelline space without entering the blastomeres. However, with the beginning of morphogenesis the dye will quickly and specifically accumlate the intestinal cells. Our observations demonstrate tissue-speclfic differentiation and presumably reflect part of the yolk accumulation process discussed ln our second abstract.

Nathaniel J Szewczyk et al. (2003) International Worm Meeting "Opposing signals from FGFR and IGFR regulate protein degradation in muscle"

Lewis A Jacobson, Nathaniel J Szewczyk, Brant K Peterson, Leah P Parkinson

[International Worm Meeting, 2003]

To identify the intramuscular signal transduction pathways that regulate proteolysis, we have used transgenic strains of C. elegans which express a soluble, enzymatically active unc-54::lacZ fusion protein in 103 body-wall and vulval muscle cells. The reporter protein is completely stable in fed wild-type animals. Reporter degradation by a pre-existing proteolytic system is triggered in fed adults when the Raf-MEK-MAPK cascade is activated in response to acute signals from LET-60 Ras or EGL-15 FGF receptor, but not from LET-23 EGFR. FGFR signals via Ras-Raf-MEK-MAPK to promote proteolysis. In contrast to degradation induced by starvation or denervation, this is not prevented by proteasome inhibitors. Protein degradation was also provoked by reduced IGFR signaling following temperature upshift of daf-2(m41ts) or age-1(hx546ts) adults or by treatment of wild-type adults with the PtdIns-3-kinase (AGE-1) inhibitor LY-290042 (LY). All of these effects can be triggered in the presence of cycloheximide and almost certainly represent non-transcriptional outputs from the signaling pathways. Two lines of evidence show that the signals are intramuscular: a) muscle-specific expression of daf-2+ prevented protein degradation in daf-2(rf) mutants; b) muscle-specific expression of age-1+ prevented degradation in an age-1(mg44) background, but did not do so in the presence of LY. LY-induced degradation was also prevented by mutations (daf-18(rf), pdk-1(gf), akt-1(gf)) that increase signal downstream of AGE-1 or by mutations (lin-45(rf), mek-2(rf), mpk-1(rf)) that block signaling downstream of Ras. Conversely, increased activity of the DAF-2 pathway (daf-18(rf)) blocked protein degradation induced by EGL-15 activation. Single mutations (let-756 or egl-17) affecting FGF-like ligands did not suppress LY-induced degradation, but there was good suppression in a let-756; egl-17 double mutant, implying that LET-756 and EGL-17 signal redundantly to activate EGL-15 FGFR. Thus, our results suggest that DAF-2 signals via AGE-1, PDK-1 and AKT-1 to inhibit LIN-45 Raf and protein degradation in muscle. They also imply that in normal adult muscle there is degradation-promoting signal from FGFR via Ras to Raf, but this signal is not passed to subsequent steps in the cascade (and protein is not degraded) unless the balancing Akt inhibition of Raf is released in consequence of low IGFR signal. This offsetting balance between two signaling pathways with distinct inputs likely enables muscle to respond to a variety of physiological changes. NJS Present address: NASA Ames Research Center, Moffett Field, CA

Rafael P Vazquez-Manrique et al. (2008) European Worm Meeting "Phospholipase C-epsilon Regulates Epidermal Morphogenesis in Caenorhabditis elegans"

Sung Ly, Rafael P Vazquez-Manrique, Aniko I Nagy, James C Legg, Olivia A M Bales, Howard A Baylis

[European Worm Meeting, 2008]

Calcium is a ubiquitous intracellular signalling molecule that controls a. wide range of cellular functions. An important means of regulating calcium. signals is through the inositol 1,4,5-trisphosphate (IP3) pathway. IP3 is. produced by the action of the enzyme phospholipase C (PLC). IP3 acts on its. ion-channel receptor, the IP3-receptor, to release Ca2+ from the. endoplasmic reticulum. It has been shown that disruption of this pathway. results in defects in several aspects of embryogenesis in Caenorhabditis. elegans (1,2). Previous work has demonstrated a role for IP3-mediated. calcium signalling in the migration of epidermal cells during embryonic. morphogenesis (1). However, the mechanism by which IP3 production is. regulated in this process is unknown. By surveying the PLC family of C.. elegans using RNAi and mutant strains, we discovered that depletion of PLC-. 1/PLC-epsilon produced substantial embryonic lethality (3). Using the epithelial. cell marker ajm-1::GFP, to follow the behaviour of epidermal cells, we. found that 96% of these arrested embryos show morphogenetic defects. These. defects include defective ventral migration and aberrant dorsal. intercalation. Using time-lapse confocal microscopy we show that the. migration of the ventral epidermal cells, especially of the leading cells,. is slower and often fails in plc-1(tm753) embryos. As a consequence plc-1. loss of function results in ruptured embryos with a Gex phenotype (gut in. the exterior) and lumpy larvae. Thus PLC-1 is involved in the regulation of. morphogenesis. Genetic studies using gain- and loss-of-function alleles of. itr-1, the gene encoding the IP3 receptor in C. elegans, demonstrate that. PLC-1 acts through ITR-1. Using RNAi and double mutants to deplete the. other PLCs in a plc-1 background, we show that PLC-3/PLC-v and EGL-8/PLC-beta. can compensate for reduced PLC-1 activity. Our work places PLC-epsilon into a. pathway controlling epidermal cell migration, thus establishing a novel. role for PLC (3).. 1.Thomas-Virnig CL, Sims PA, Simske JS, Hardin J. (2004) The inositol. 1,4,5-trisphosphate receptor regulates epidermal cell migration in. Caenorhabditis elegans. Curr Biol.14:1882-7.. 2.Walker DS, Gower NJ, Ly S, Bradley GL, Baylis HA. (2002) Regulated. disruption of inositol 1,4,5-trisphosphate signaling in Caenorhabditis. elegans reveals new functions in feeding and embryogenesis. Mol Biol. Cell. 13:1329-37.. 3.Vazquez-Manrique RP, Nagy AI, Legg JC, Bales O AM, Ly S, Baylis HA.. (2008) Phospholipase C-epsilon Regulates Epidermal Morphogenesis in. Caenorhabditis elegans.PLOS Genetics, in press

Cheon, YongJin et al. (2021) International Worm Meeting "ascr#3 imprinting is mediated by chromatin remodeling"

Cheon, YongJin, Hwangbo, Semin, Kim, Kyuhyung

[International Worm Meeting, 2021]

Imprinting is a form of long-lasting and robust memory which has been described in many animals including human. C. elegans, early experience of ascaroside#3 pheromone (ascr#3, C9, asc-deltaC9) enhances ascr#3 avoidance of adult hermaphrodites via the functional modification of the ascr#3 avoidance circuit. Upregulation of odr-2 Ly-6-related GPI-linked signaling gene expression in the SMB motor neuron is required for ascr#3 imprinting (Hong et al., 2017). However, circuit and molecular mechanisms underlying imprinting need to be further investigated. Here we show that neuronal chromatin remodeling mechanism may play a critical role for ascr#3 imprinting. We first performed candidate gene search including set-2 sSET1/MLL histone H3K4 methyltransferase gene. We found that ascr#3 imprinting is abolished in set-2 mutants. We also found that, whereas expression of set-2 under the control of own promoter did not rescue imprinting defect of set-2 mutants, these defects were partially restored upon expression of set-2 cDNA under the control of pan-neuronal promoter. We are currently performing additional rescue experiment. We are also observing odr-2 expression level and performing SMB Ca2+ imaging in set-2 mutant background. This study will allow us to define a common logic for long-lasting memory formation and other forms of behavioral plasticity.

Olivia Am Bales et al. (2005) International Worm Meeting "Defining IP3 signals in the C. elegans pharynx."

Olivia Am Bales, Howard A Baylis

[International Worm Meeting, 2005]

Worms show an increased pharyngeal pumping rate on exposure to food, and this increase requires signalling by the second messenger inositol 1,4,5-trisphosphate (IP3) through the IP3 receptor itr-1 1. We are using complementary techniques to identify the signalling components upstream of IP3 production and to observe the spatiotemporal nature of the IP3 signal in the pharynx. Phospholipase C hydrolyses the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to generate IP3, and the C. elegans phospholipase C genes plc-3 and plc-4 are expressed in pharyngeal muscle. RNA interference and analysis of a loss-of-function mutant suggest a role for plc-3 in the upregulation of pharyngeal pumping on food. We are also developing a system for visualising IP3 signals in the pharynxes of intact worms, using a fusion protein of GFP and the pleckstrin homology (PH) domain. Translocation of this protein can be used to follow the production of IP3 2. We are currently investigating the movements of the PH-GFP fusion in wild-type and mutant worms following stimulation by the muscarinic agonist arecoline.1. Walker, D. S., Gower, N. J., Ly, S., Bradley, G. L., and Baylis, H. A. (2002). Regulated disruption of inositol 1,4,5-trisphosphate signaling in Caenorhabditis elegans reveals new functions in feeding and embryogenesis. Mol Biol Cell 13, 1329-1337.2. Varnai, P., and Balla, T. (1998). Visualization of phosphoinositides that bind pleckstrin homology domains: calcium- and agonist-induced dynamic changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools. J Cell Biol 143, 501-510.

Janzen, Laine et al. (2011) International Worm Meeting "Forward screen for mutants lacking a food block on olfactory adaptation."

L'Etoile, Noelle, Cha, Amanda, Ghafari, Ghazal, Llamas, Sara, Young, Jared, Janzen, Laine, Stinson, Pilar, Summers, Stephanie

[International Worm Meeting, 2011]

Context cues play a role in compulsive and addictive behaviors by endowing accompanying stimuli with increased salience. For example, olfactory cues such as food odors accompany the reinforcing stimulus of the food itself, and thus stimulate cravings for the reinforcing stimulus. We suggest important modulation of odor processing may occur in olfactory sensory neurons, and indeed recent studies have indicated that responses to odors may be modified early in the olfactory pathway (1,2). Olfactory adaptation is a basic form of learning that causes desensitization of an organism to an odorant after a prolonged period of exposure. Studies have shown that adaptation to an odorant can be repressed by the presence of food in the model organism C.elegans (3,4). This block of adaptation involves inhibition of changes in the primary sensory neuron which are associated with olfactory adaptation. As part of a larger project to study the molecular, cell biological, and circuit level mechanisms by which food-derived signals affect odor signaling and long-term plasticity in the model organism C.elegans, this project will attempt to identify new genes involved in the inhibition of long-term neuronal plasticity by food signaling in the sensory neuron (AWC). To identify new genes involved in the food block on adaptation, we are using an unbiased forward genetic screen to isolate mutants. This foundational project will contribute to a larger endeavor to reveal how rewarding stimuli alter signal processing within the primary sensory neuron and may inspire therapies for controlling debilitating compulsive behaviors, such as over eating or drug addiction. 1. Li W, Howard JD, Parrish TB, Gottfried JA. Aversive learning enhances perceptual and cortical discrimination of indiscriminable odor cues. Science. 2008 Mar 28;319(5871):1842-5. 2. Doucette W, Gire DH, Whitesell J, Carmean V, Lucero MT, Restrepo D. Associative Cortex Features in the First Olfactory Brain Relay Station, Neuron. 2011 Mar;69(6):1176-1187. 3. L'Etoile ND, Coburn CM, Eastham J, Kistler A, Gallegos G, Bargmann CI. The cyclic GMP-dependent protein kinase EGL-4 regulates olfactory adaptation in C. elegans. Neuron. 2002 Dec 19;36(6):1079-89. 4. Colbert HA, Bargmann CI. Environmental signals modulate olfactory acuity, discrimination, and memory in Caenorhabditis elegans. Learn Mem. 1997 Jul-Aug;4(2):179-91.