Yoshida, K. et al. (2019) International Worm Meeting "Identification of a novel RNAi-related factor that promotes intercellular transport of double-stranded RNA."

Yoshida, K., Mitani, S., Yoshina, S., Suehiro, Y.

[International Worm Meeting, 2019]

During the process of systemic RNA interference (RNAi), consisting of secretion from donner cells and uptake into recipient cells of double-stranded RNA (dsRNA), membrane trafficking is thought to play important roles. However, it is not well understood which membrane trafficking pathways are involved in the transport of dsRNA. To identify the secretory pathway of dsRNA in systemic RNAi, we screened for mutants that show defects in RNAi triggered by ingested dsRNA under the genetic background of double mutant for rab-3 and aex-6/rab-27, which encode Rab-GTPases regulating the trafficking pathway for secretion. We identified two independent mutations in the Y39A3CL.1 gene that are partially deficient in the feeding RNAi. Another mutant carrying a Y39A3CL.1 deletion also showed RNAi resistance both in somatic cells and in the germline. Y39A3CL.1 did not genetically interact with rab-3 and aex-6 double mutation whereas it was originally identified by mutagenizing the double mutant. Y39A3CL.1 encodes a protein with no known domains and has no homologs outside of nematodes. Y39A3CL.1 is expressed in various tissue including hypodermis, pharynx and intestine. Tissue-specific rescue experiments revealed that Y39A3CL.1 can act in a cell non-autonomous manner during systemic RNAi. We examined subcellular localization of Y39A3CL.1 fused with RFP and found that it co-localizes with late endosome and lysosome markers. These results suggest that Y39A3CL.1 is a novel RNAi-related factor which may act in an endomembrane-trafficking pathway and promote intercellular transport of dsRNA.

Yoshida, K. et al. (2011) International Worm Meeting "Olfactory preference switch depending on odor concentration is mediated by combinatorial change of acting sensory neurons."

Hirotsu, T., Oda, S., Tagawa, T., Yoshida, K., Ishihara, T., Iino, Y.

[International Worm Meeting, 2011]

Many organisms exhibit olfactory preference towards most odorants. It is empirically known that the perceived quality of an odorant changes depending on its concentration in some organisms including humans. However, the neuronal basis of the preference change depending on odor concentration is still elusive. Here we show C. elegans also shows changes of olfactory preference depending on odor concentration. When concentrations of various attractive odorants such as isoamyl alcohol are increased, animals become to show avoidance of them. Behavioral analyses and computer simulation revealed that the behavioral change depending on concentration of an odorant is mainly mediated by one of two known behavioral strategies, klinokinesis (the pirouette mechanism). Namely, the dependence of the frequency of pirouette behavior on temporal changes in odor concentration is reversed in different concentration ranges. On the other hand, the other behavioral strategy klinotaxis (the weathervane mechanism) is employed in the direction to drive attraction to the odorant regardless of its concentration. We next examined what molecules mediate these behaviors, and found that ODR-3 G protein a subunit mediates klinokinesis and klinotaxis in the concentration-dependent chemotaxis to isoamyl alcohol. Moreover, ODR-3 transmits the olfactory signaling depending on isoamyl alcohol concentration by acting in different sensory neurons. We further show that distinct combinations of sensory neurons function at different concentrations of isoamyl alcohol: AWC sensory neurons are known to mediate attraction to lower concentration of isoamyl alcohol, whereas ASH, AWB and ADL sensory neurons are required for avoidance of higher concentration of it. Calcium imaging revealed that activity patterns of these sensory neurons dramatically change depending on isoamyl alcohol concentration: the higher concentration of isoamyl alcohol fails to elicit responses of AWC, which strongly responds to lower concentration of the odorant, and elicits particular activity patterns of AWB and ASH. Hence, our study shows that the information processing which regulates olfactory preference change depending on odor concentration is clearly explained by the labeled-line theory.

Yoshida K et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Neural and Behavioral Mechanisms of Response to an Odorant Depending on its Concentration"

Ishihara T, Yoshida K, Tagawa T, Iino Y, Oda S, Hirotsu T

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

The perceived quality of an odorant can differ with a change in its concentration in many species. In C. elegans, it was reported that several odorants such as benzaldehyde and 2,4,5-trimethylthiazole, act as attractants at low concentrations, but as repellents at high concentrations. We found that this dose-dependent behavioral change was also observed in chemotaxis to all the odorants tested (isoamyl alcohol, diacetyl, and 2,3-pentanedione). To examine the underlying neural circuit, we performed laser ablation of individual neurons and found that avoidance of high concentration of isoamyl alcohol is regulated by multiple sensory neurons. Moreover, we identified some interneurons which positively mediate this avoidance. It was previously reported that AWC sensory neurons regulate attraction to diluted isoamyl alcohol and are activated by the removal of isoamyl alcohol. Our results of calcium imaging revealed that AWB and ASH sensory neurons, which are both known to mediate an avoidance behavior, respond to the removal and addition of isoamyl alcohol, respectively. Recently, we reported that worms employ two strategies (the pirouette and weathervane strategies) for efficient chemotaxis. To investigate the behavioral mechanisms of avoidance of isoamyl alcohol at the high concentration, we analyzed this behavior by using multi-worm tracking system. Though the pirouette mechanism was mainly used to regulate this avoidance behavior, interestingly, the weathervane mechanism was employed to curve toward isoamyl alcohol. Computer simulation showed that behaviors of model animals using both strategies are quantitatively similar to those of real worms. We also looked for molecules involved in avoidance of isoamyl alcohol at the high concentration, and found that mutants of odr-3, which encodes a G-protein alpha subunit, show a severe defect in this avoidance. Furthermore, cell-specific rescue experiments suggested that the function of ODR-3 in AWB sensory neurons is important for avoidance of isoamyl alcohol.

Chun-Yi Huang et al. (2007) International Worm Meeting "eif-3.K is a pro-apoptotic gene in C. elegans."

Jia-Yun Chen, Yi-Chun Wu, Chun-Yi Huang

[International Worm Meeting, 2007]

Programmed cell death (or apoptosis) is an important feature of C. elegans development. Previous studies have identified pro-apoptotic genes egl-1, ced-3 and ced-4 and anti-apoptotic genes ced-9 and icd-1 that control programmed cell death.. We have identified and characterized a novel pro-apoptotic gene eif-3.K. Loss-of-function by mutation or RNAi inactivation in eif-3.K resulted in a decrease of cell corpses, whereas heatshock-induced over-expression of eif-3.K weakly but significantly increased cell corpses. Interestingly, the eif-3.K mutation partially suppressed ectopic cell deaths caused by over-expression of egl-1 or ced-4. This result suggests that eif-3.K may act downstream of or in parallel to egl-1 and ced-4 in the programmed cell death pathway. Using a cell-specific promoter to express eif-3.k in touch neurons, we showed that eif-3.K likely promoted cell death in a cell-autonomous manner. To further explore EIF-3.K function, we generated antibodies against bacterially expressed EIF-3.K protein. We found that EIF-3.K was ubiquitously expressed during embryogenesis and localized to the cytoplasm. As human eif-3.K can functionally substitute C. elegans eif-3.K in an eif-3.K mutant, the function of eif-3.K in apoptosis is likely conserved in evolution.

Chun-Yi Huang et al. (2006) East Asia C. elegans Meeting "eif-3.K is a pro-apoptotic gene in C. elegans"

Teng-Wei Huang, Chun-Yi Huang

[East Asia C. elegans Meeting, 2006]

Programmed cell death or apoptosis is an important feature during C. elegans development. The pro-apoptotic genes egl-1, ced-4 and ced-3 are required for the execution of cell death. We have identified and characterized a novel pro-apoptotic gene eif-3.K. A loss-of-function mutation or inactivation by RNA interference in eif-3.K resulted in a reduction of cell corpse number during embryogenesis, whereas heatshock-induced over-expression of eif-3.K weakly but significantly promoted programmed cell death. In addition, eif-3.K mutation partially suppressed ectopic cell deaths caused by over-expression of egl-1 and ced-4. This result suggests that eif-3.K may act downstream of or in parallel to egl-1 and ced-4 in the genetic pathway during programmed cell death. Using a cell-specific promoter we showed that eif-3.K likely promoted cell death in a cell-autonomous manner. We generated antibodies against bacterially expressed EIF-3.K protein. The immunostaining result showed that EIF-3.K was ubiquitously expressed during embryogenesis and localized to the cytoplasm. To better understand the cell-death defect of eif-3.K mutants, we are currently performing a 4D microscopic analysis of the cell death process in wild-type and eif-3.K mutants.

Kalogeropoulou, E. et al. (2017) International Worm Meeting "Caenorhabditis elegans exhibits an increase in the probability of pirouettes and head swing amplitude in the absence of the SAA class of neurons."

Hope, I.A., Cohen, N., Kalogeropoulou, E.

[International Worm Meeting, 2017]

Many of the behaviours of C. elegans are mediated by the worm's ability to orient itself towards chemical gradients or other sensory cues. Two mechanisms of orientation have been observed in the worm: pirouettes and steering. Past studies have identified a number of neuron classes that contribute to navigation by linking neuronal laser ablations and observations of mutants to changes in body postures and locomotion statistics1,2. Recently, optogenetic manipulation has also shed light on the neuronal control of the worm's chemotactic behaviour3. SMB motor neurons have been postulated to be part of the navigation circuit. We have generated a chromosomally-integrated transgenic line (UL4230) where the SMBs are genetically ablated in early larvae. The line demonstrates a loopy phenotype similar to that previously observed following laser-ablation of SMBs1. We have also targeted another class of neuron, located close to and highly connected via synapses to the SMBs but not previously explored: the SAA interneurons. We initially used expression of GFP to confirm that the selected promoters drove expression in the location originally described. A combination of lad-2 and unc-42 promoters was used to genetically ablate the SAAs by targeted expression of the worm's caspase, as encoded by two distinct parts of the ced-3 gene, such that the intact enzyme is produced only in these neurons specifically4. Absence of GFP expression confirmed the specific and targeted ablation of the SAAs. The generated strains, with SMBs or SAAs ablated (UL4230 and UL4207), demonstrated a phenotype different to that of N2 in both spontaneous and evoked locomotion. Locomotion was evoked using a radial gradient of NH4Cl. Initial results suggest that both SMBs and SAAs suppress the probability of pirouettes and decrease the amplitude of sinusoidal undulations. Additional experiments are underway to explore the contributions of SAA and SMB, separately and together, to undulations and steering, and to link those to the pirouette initiation pathway. 1. Gray J. M., et al., (2005). A circuit for navigation in Caenorhabditis elegans. Proc. Natl. Acad. Sci. U.S.A, 102(9). 2. Iino Y., and Yoshida K. (2009). Parallel use of two behavioral mechanisms for chemotaxis in Caenorhabditis elegans. Journal of Neuroscience, 29(17). 3. Kocabas A., et al., (2012). Controlling interneuron activity in Caenorhabditis elegans to evoke chemotactic behaviour. Nature, 490(7419). 4. Chelur D. S., and Chalfie M. (2007). Targeted cell killing by reconstituted caspases. Proc. Natl. Acad. Sci., 104(7).

Johnson, Christina K. et al. (2019) International Worm Meeting "Requirement of glial Na+/K+-ATPase in touch sensation highlights ionic and metabolic link between glia and touch neurons in C. elegans."

Johnson, Christina K., Bianchi, Laura, Wang, Ying, Fernandez Abascal, Jesus

[International Worm Meeting, 2019]

Isolated microenvironments, such as the tripartite synapse, where the concentration of ions is regulated independently from the surrounding tissues, exist throughout the nervous system, including in mechanoreceptors. Modulation of the ionic composition of these microenvironments has been suggested to be achieved by glia and other accessory cells. However, the molecular mechanisms of ionic regulation and effects on neuronal output and animal behavior are poorly understood. Using the model organism C. elegans, our lab published that Na+ channels of the DEG/ENaC family expressed in glia control neuronal Ca2+ transients and animal behavior in response to sensory stimuli. DEG/ENaC Na+ channels are known to establish a favorable driving force for K+ excretion, which occurs via inward rectifier K+ channels, in epithelial tissues across species. We hypothesized that a similar mechanism exists in the nervous system. Using molecular, genetic, in vivo imaging, and behavioral approaches, we showed that expression in glia of inward rectifier K+ channels and cationic channels rescues the sensory deficits caused by knock-out of glial DEG/ENaCs without disrupting neuronal morphology, supporting our hypothesis. Based on this model, Na+/K+-ATPases are also needed to maintain ionic concentrations following influx of Na+ and excretion of K+. We show here that, in addition to glial Na+ and K+ channels, two specific glial Na+/K+-ATPases, EAT-6 and CATP-1, are needed for touch sensation and that their requirement can be bypassed by a high glucose diet. The effect of glucose is dependent on ATP binding capability of the pump, translation, transcription, and the activity of CATP-2, a third Na+/K+-ATPase ?-subunit. Taken together, our results support metabolic and ionic cooperation between glia and neurons in C. elegans mechanosensors, a mechanism that is essential to regulating neuronal output and may be conserved across species.

Stefano Berri et al. (2009) European Worm Neurobiology Meeting "An integrated model of C.elegans forward locomotion."

Netta Cohen, Ian A Hope, Jordan H Boyle, Stefano Berri

[European Worm Neurobiology Meeting, 2009]

We study C. elegans locomotion in a variety of homogeneous as well as complex environments, with an aim to achieve an integrated model of the neural control of locomotion. We began by studying the transition in forward locomotion from swimming (in liquid) to crawling (in a stiff gel). By analysing the worm''s locomotion in a range of media with increasing gel concentration, we demonstrated that swimming and crawling are achieved by modulation of a single gait[1]. This finding suggests that a single neural mechanism underlies this entire range of observed behaviours. It further indicates that the physics of the environment plays an important role in modulating the worm''s locomotion. We therefore developed an integrated model of the worm''s forward locomotion that consists of a ventral cord nervous system, muscles, a body and a physical environment. The neural model consists of excitatory B-like neurons driven by proprioceptive (stretch receptor) input as well as inhibition from D-type neurons. The integrated model can generate and propagate coordinated undulations. In the model, the entire swim-crawl transition can be achieved by a single (i.e., fixed-parameter) model worm, embedded in a range of environments (with different resistive drag properties). Model predictions and extensions: 1) The model predicts a natural frequency-wavelength relationship in freely locomoting worms. Simulation experiments of locomotion in microfluidic "artificial dirt" chips and irregular granular media shed light on the behaviour of the worm and provide an interesting way to decouple the undulation frequency from wavelength along the body. 2) The model predicts that D-class GABAergic motoneurons have an important role in forward locomotion, with an effect more clearly visible in water than on agar. Our experiments confirm this prediction: mutants in the GABA pathway such as unc-25(e156), unc-30(e191), unc-46(e177), unc-47(e307) and unc-49(e382) all exhibit severely uncoordinated phenotype in water but nearly wild type behaviour on agar. 3) Finally, a model of the neural control of forward locomotion lends itself naturally to a study of turning mechanisms. Specifically here we focus on the gentle turns recently implicated in chemotactic response (the "weathervane" strategy[2]). We present model simulations that shed light on the respective contributions of the head versus the rest of the body in initiating and controlling dorso-ventral asymmetries in the body bending that are required to perform a change of direction. [1] Berri S, Boyle JH, Tassieri M, Hope IA and Cohen N. 2009. "Forward locomotion of the nematode C. elegans is achieved through modulation of a single gait." HFSP journal, 3(3):186-193. [2] Iino Y. and Yoshida K. 2009. "Parallel use of two behavioral mechanisms for chemotaxis in Caenorhabditis elegans". Journal of Neuroscience, 29(17):5370-5380

Miyanishi, Yosuke et al. (2011) International Worm Meeting "Functional imaging of neuronal activity for 2-nonanone stimulation."

Nakai, Junichi, Kimura, Kotaro, Miyanishi, Yosuke

[International Worm Meeting, 2011]

To better understand the neural basis that regulates a worm's sensory behavior and its modulation by learning, we are studying avoidance behavioral responses to 2-nonanone. We previously reported that the avoidance behavior to 2-nonanone is enhanced, rather than reduced, after preexposure to the odor, and this enhancement is acquired as a non-associative dopamine-dependent learning (Kimura et al., J. Neurosci., 2010; Fujita and Kimura, this abstract). In addition, we observed that worms respond to a spatial gradient of 2-nonanone (Yamazoe and Kimura, CE Neuro, 2010), which cannot be simply explained by the pirouette or weathervane strategies. 2-nonanone is mainly sensed by the AWB neurons, which have been shown to exhibit odor-OFF response in aqueous step stimulation with 2-nonanone (Troemel et al., Cell 1997; Ha et al., Neuron 2010). To understand how the neuronal circuits of worms regulate the characteristic 2-nonanone behavioral response, we are monitoring calcium changes in the AWB and downstream neurons using G-CaMP 4 (Shindo et al., PLoS ONE, 2010). We thank Drs. S. Oda, K. Yoshida, and Y. Iino (U. Tokyo) for suggestions on microfluidics; M. Hendricks and Y. Zhang (Harvard) for aqueous 2-nonanone stimulation; and E. Busch and M. de Bono (MRC) for gaseous microfluidic stimulation.

Maelle Jospin et al. (2001) International C. elegans Meeting "Basic electrophysiological properties of body wall muscle from the Nematode C elegans."

Maelle Jospin, Laurent Segalat, Bruno Allard

[International C. elegans Meeting, 2001]

Electrophysiological properties of striated muscle cells were investigated with the patch clamp technique in the Nematode C elegans . Worms were immobilised with cyanoacrylate glue and longitudinally incised using a tungsten rod sharpened by electrolysis. Recording pipettes were sealed on GFP-expressing body wall muscle cells. In the whole cell configuration, under current clamp conditions, in the presence of Ascaris medium in the bath and K-rich solution in the pipette, no action potential could be induced in response to current injection. Under voltage clamp control and in the same ionic conditions, depolarizations above -30 mV from a holding potential of -70 mV gave rise to outward K currents. Outward K currents resulted from two components, one fast inactivating component blocked by 4-aminopyridine, one delayed sustained component blocked by tetraethylammonium. In the presence of both blockers, an inward Ca current was revealed and inhibited by cadmium. Single channel recording using the inside-out configuration revealed the existence of a Ca-activated Cl channel and a Ca-activated K channel. Single channel experiments are currently performed to characterise voltage-gated conductances at the unitary level.