Helms, Stephen J et al. (2013) International Worm Meeting "A Common Behavioral Model Underlies the Motility of a Diverse Set of Nematodes."

Avery, Leon, Helms, Stephen J, Shimizu, Thomas S, Stephens, Greg J

[International Worm Meeting, 2013]

Animal behavior emerges from many layers of biological organization-from molecular signaling pathways and neuronal networks to mechanical outputs of muscles. Naively, the large number of interconnected variables at each of these layers would seem to imply dynamics that are complex and hard to control or even tinker with. Yet, for organisms to survive in a competitive, ever-changing environment, behavior must readily adapt. We have quantitatively characterized motile behavior in a diverse set of nematodes spanning the lab strain C. elegans N2 to wild strains and distant species. Using high-resolution imaging, we measured the centroid trajectory and body shape dynamics of individual worms moving on agar plates surrounded by a repellant boundary. We found that a simple physical model accounting for variable speed, stochastic reversal events, and an anomalously diffusing bearing can describe all the tested species. Furthermore, these behaviors are driven by conserved patterns in the postures adopted by the worms. Finally, we found that the behavioral parameters varied over time, among individuals, and across strains and species in a correlated way that is consistent with a trade-off between exploratory and exploitative behavior. This suggests a surprisingly simple underlying mechanism controlling variation in motile behavior that matches likely relevant ecological constraints.

Helms, Stephen et al. (2015) International Worm Meeting "A Roaming-Dwelling Axis Dominates Variation in Motile Behavior Across Nematode Species."

Shimizu, Tom, Helms, Stephen, Carlos-Costa, Antonio, Avery, Leon, Stephens, Greg

[International Worm Meeting, 2015]

A quantitative understanding of organism-level behavior requires predictive models that can capture the richness of behavioral phenotypes, yet are simple enough to extract functional and mechanistic insights. A key challenge is to develop and interpret such models in an unbiased manner. Behavior combines elements of randomness and determinism, thus requiring extensive data sets from well-controlled laboratory experiments to avoid biases in sampling its statistical repertoire. On the other hand, the biological significance of the extracted quantitative features needs to be interpreted in the broader context of its ecology and physiology. We developed an experimental setup and analysis protocol that allows us to extract at high throughput the translational trajectories and body postures of many individual nematode worms over extended times. We applied this system to characterize a panel of strains originating from diverse ecological contexts, including the canonical lab strain C. elegans N2, additional wild isolates of C. elegans, and other species spanning a large portion of the nematode phylum. From this rich quantitative dataset, we found that trajectory statistics of all tested worms could be described by a common random-walk model with independent dynamics in the speed and bearing. Differences across strains and species, as well as between individuals within each strain, are captured by variation in the seven parameters that completely describe the model. Cross-species comparisons of extracted parameters revealed that the principal mode of variation is in the degree of spatial exploration (corresponding to 'roaming' and 'dwelling' behaviors). We are now extending this work to include the higher-resolution dynamics of the worm's body postures. Our approach combining data-driven modeling with cross-species comparisons provides an objective means of identifying biologically relevant patterns of behavioral variation and simple statistical models that will be instrumental for future mechanistic studies of behavioral control.

Helms, Stephen et al. (2015) International Worm Meeting "Quantitative Behavioral Measurements of Individual Aging Worms Reveal Lifespan-Altering Insulin Pathway Mutants Do Not Affect Healthspan."

Lezzerini, Marco, Lohrmann, Esther, Koers, Jana, Shimizu, Tom, Stephens, Greg, Helms, Stephen, Budovskaya, Yelena

[International Worm Meeting, 2015]

Animals exhibit complex behaviors that emerge from the coordinated function across multiple scales of an organism: from genetics to neurons and physiology. Therefore, quantitative behavioral measurements can provide insight into the overall 'health' of an organism. We have developed a high-throughput imaging experiment to measure the motility and posture of individual worms on agar with food over their entire adult life. These data allow for a rich statistical characterization of the behavior of individual worms and how such behavior changes over time. We used this new imaging experiment to compare behavioral changes during aging in wild-type and the classic lifespan-affecting insulin pathway mutations: daf-2 (CF1580, twice as long), daf-16 (CF1874, slightly shorter), and daf-2/daf-16 (CF1588, slightly shorter). Each mutation was backcrossed into the sod-3::GFP reporter strain (CF1553) to ensure no spurious mutations had accumulated. For each strain, we performed two independent experiments assaying 24 worms for an hour 16 times over 31 days. In related work, we developed a parameterized model of motility. Using this model, we found that aging was associated with a decrease in exploratory motility and a progressive slowing of behavior. While we did observe the expected changes in lifespan, all of the strains showed a similar and reproducible decline in behavior as the worms aged. A number of recent studies have underscored the view that changes in lifespan are not necessarily associated with changes in aging-related functional decline ('healthspan'). Our result confirms that the increased lifespan in daf-2 is not accompanied by an increase in healthspan, and provides a detailed view of how behavior changes with age. Furthermore, this work demonstrates the power of our experimental system and automated analysis for rapid behavioral phenotyping.

Rachel McMullan et al. (2006) European Worm Meeting "Rho is a Pre-Synaptic Activator of Neurotransmitter Release at Pre-Existing Synapses in C.elegans"

Rachel McMullan, Emma Hiley, Paul Morrison, Stephen J. Nurrish

[European Worm Meeting, 2006]

Rachel McMullan, Emma Hiley, Paul Morrison and Stephen J. Nurrish. Rho GTPases have important roles in neuronal development but their function in adult neurons is less well understood. We demonstrate that pre-synaptic changes in Rho activity at C.elegans neuromuscular junctions can radically change animal behaviour via modulation of two separate pathways. In one, pre-synaptic Rho increases acetylcholine (ACh) release by stimulating the accumulation of diacylglycerol (DAG) and the DAG-binding protein UNC-13 at sites of neurotransmitter release; this pathway requires binding of Rho to the DAG kinase DGK-1. A second DGK-1-independent mechanism is revealed by the ability of a Rho inhibitor (C3 transferase) to decrease levels of release even in the absence of DGK-1; this pathway is independent of UNC-13 accumulation at release sites. We do not detect any Rho induced changes in neuronal morphology or synapse number, thus Rho facilitates synaptic transmission by a novel mechanism. Surprisingly, many commonly available human RhoA constructs contain an uncharacterised mutation that severely reduces binding of RhoA to DAG kinase. Thus a role for RhoA in controlling DAG levels has not been previously appreciated.

de Vries CJ et al. (1998) European Worm Meeting "A cytosolic dynein heavy chain is required for sensory neuron structure and function."

de Vries CJ, Wicks SR, Plasterk RHA

[European Worm Meeting, 1998]

We have developed two new assays of nematode chemotaxis (see abstract Stephen Wicks). The first gene identified and cloned using these assays was a cytosolic isoform (1B subfamily) of the dynein heavy chain. Complementation studies show that this dynein heavy chain is the CHE-3 protein. Previously, this isoform of the dynein heavy chain was shown to be expressed during cilia outgrowth in sea urchins (Gibbons, 1994), and involved in the organisation of the Golgi-apparatus in non-ciliated human cell lines (Vaisberg, 1996). GFP expression studies indicate that che-3 is expressed exclusively in all ciliated sensory neurons in the worm. The highest levels of che-3 expression are correlated with outgrowth of cilia. To visualise the structure of ciliated endings we used two GFP markers of amphid and phasmid integrity (GPA-13::GFP and GPA-15::GFP, as integrated transgenes, gifts of Gert Jansen). These markers indicate that the amphid and phasmid cilia are malformed in the worm, and that the degree of disorganisation appears to increase as the animal ages. These results suggest that dynein heavy chain has a function in cilia outgrowth in C. elegans, but has no role in the organisation of the Golgi apparatus in the worm. Gibbons, B.H., Asai, D.J., Wen-Jing, Y., Hays, T.S., and Gibbons, I.R. (1994). Phylogeny and expression of axonemal and cytoplasmic dynein genes in sea urchins. Mol. Biol. Cell 5, 57-70. Vaisberg, E.A., Grissom, P.M., and McIntosh, J.R. (1996). Mammalian cells express three distinct dynein heavy chains that are localized to different cytoplasmic organelles. J. Cell Biol. 133, 831-842.

Ralph Menzel et al. (2006) European Worm Meeting "PCB52 Induced Gene Expression in the Nematode Caenorhabditis elegans"

Jana Kulas, Stephen Sturzenbaum, Shuang Li, Hui Ling Yeo, Ralph Menzel

[European Worm Meeting, 2006]

Ralph Menzel1, Jana Kulas1, Hui Ling Yeo1, Shuang Li1, and Stephen Strzenbaum2. Polychlorinated biphenyls (PCBs) are ubiquitous organic chemicals which can affect numerous biological responses. Because PCBs are toxic to man, recalcitrance to degradation and persist in the environment, they are listed in the inventory of priority pollutants compiled by numerous Environmental Protection Agencies.. Here we present an investigation of PCB52, an ortho-substituted, non-coplanar 2,2,5,5-tetrachlorbiphenyl. PCB52 affected the reproductive capacity of C. elegans in a strict concentration dependent manner, thereby highlighting its toxicological significance. Based on the calculated EC20 (5 mg/L), whole genomic DNA microarray experiments were performed to identify differentially expressed genes. Using a two-fold increase/decrease cut-off, 1158 genes were up-regulated and 560 down-regulated by PCB52. Up-regulated genes constituted family members of small heat shock proteins, chemoreceptors, nuclear hormone receptors, lipases as well as cytochromes P450 (CYP) and short-chain dehydrogenases/reductases (SDR), which are known to be involved in the biotransformation of persistent organic pollutants. Significant enriched classes of down-regulated genes are involved in organelle biogenesis, cell integrity and further general physiological processes, such as macromolecule metabolism and nucleotide binding.. The induction of five highly differentially regulated gene classes was confirmed by semi-quantitative RT-PCR experiments. Using a multiple RNAi approach the consequences of gene knockdown of single genes as well as entire gene subfamilies was determined. The efficiency of RNAi was proven by RT-PCR. Whilst GST and SDR gene expression knockdown intensify the toxicity of PCB52 (reflected in a reduced reproductive output) the knockdown of several CYP forms increased the reproductive capacity. This may indicate that the lack of CYP gene expression prevents the generation of toxic metabolites.

Thiele TR et al. (2000) West Coast Worm Meeting "Mutants with altered sensitivity to the effects of ethanol on locomotion"

Eastman CL, McIntire SL, Thiele TR, Davies AG

[West Coast Worm Meeting, 2000]

Ethanol is reported to have multiple targets in the nervous system, a factor that has complicated efforts to understand the actions of ethanol that bring about behavioral effects in mammalian systems. We are using C. elegans to confirm suspected targets of ethanol and identify new targets and pathways that can be affected by ethanol to produce behavioral changes. We have concentrated on the effects of ethanol on locomotion. With increasing doses of ethanol in a plate assay, wild-type animals display a decrease in activity, velocity and amplitude of the body waveform. Non-saturating screens for mutants with altered sensitivity to ethanol for locomotion have produced eight mutants with increased resistance to ethanol and four mutants with increased sensitivity to ethanol. We have characterized the locomotion, egg laying and pharyngeal pumping behaviors of these mutants in the presence and absence of ethanol and examined nervous system function by pharmacological analysis. Mapping and cloning of the genes mutated in these strains is underway using positioned Tc1 polymorphisms or single nucleotide polymorphisms that result in alterations to restriction enzyme sites (thanks to Stephen Wicks for unpublished data). We have identified one of the resistance genes as the calcium-activated potassium channel gene slo-1. Potassium channels of the slo class are thought to regulate the excitability of neurons. There is in vitro evidence for a direct activating interaction of ethanol on channels of this class derived from mammalian systems (reviewed by Dopico et al. 1999, Neurochem. Int. 35:103-106) raising the possibility that we may have identified a conserved direct target of ethanol in the nervous system of the worm.

M Wayne Davis et al. (2005) International Worm Meeting "High-throughput SNP mapping primers and techniques"

Erik M Jorgensen, Tracey Harrach, Marc Hammarlund, Patrick Hullett, M Wayne Davis, Shawn Olsen

[International Worm Meeting, 2005]

Single nucleotide polymorphism (SNP) mapping against CB4856 has quickly become an indispensable technique in the worm field since its introduction by Stephen Wicks et al. in 2001 (Nat Genet 2001 28: 160-164). Because SNPs are present every few thousand base pairs, SNP mapping allows mapping to extremely narrow intervals. Because mapping is done in an essentially wild-type background, very subtle or complex phenotypes can be mapped that could not be mapped using visible mutations. Typically, SNPs that alter a restriction enzyme recognition site (snip-SNPs) have been detected by running digested PCR products on agarose gels. This technique is reliable and readily accessible to any molecular biology lab; however, it requires substantial hands-on time for each reaction, as each SNP typically requires different PCR cycling and restriction digestion conditions. A number of methods have been published that provide automated analysis of SNP genotypes, including florescence polarization (Swan et al. 2002 Genome Res 12: 1100-1105) or capillary electrophoresis (Zipperlen et al. 2005 Genome Biol 6: R19). Although these methods have the advantage of reducing hands-on time for determining SNP genotypes, they require investment in expensive equipment that is not common in many molecular biology labs. In order to provide a more efficient detection protocol for snip-SNPs, we have developed a set of 48 primer pairs that detect SNPs evenly spaced across the C. elegans genome, and that work under identical PCR and restriction digestion conditions. We present a scheme using these reagents to quickly and reliably map mutations using high-throughput equipment and techniques. We have used our SNP mapping techniques to map many phenotypically subtle or genetically complex mutants, including a subtle behavioral defect resulting from three independent mutations. In fact our techniques are simple and relatively inexpensive enough to have been used successfully by undergraduates in a teaching laboratory setting.

Suzumi M. Tokuoka et al. (2006) European Worm Meeting "Valproic Acid Acts on Both IP3 and DAG Regulated Behaviours in C. elegans"

Stephen Nurrish, Suzumi M. Tokuoka

[European Worm Meeting, 2006]

Suzumi M. Tokuoka and Stephen Nurrish. Phosphatidylinositol-4,5-bisphosphate (PIP2) is hydrolysed by Phospholipase C into Inositol tri phosphate (IP3) and diacylglycerol. Recent work has suggested that defects in inositol phophate signalling may underlie behavioural defects such as bi-polar disorder (manis depression). In support of this model commonly used drugs to treat bipolar disorder, such as Lithium and Valproic acid (VPA) (Nature 417:292 2002). Lithium is known to inhibit enzymes involved in recycling of inositol phosphates but the targets of VPA are unclear. We are searching for VPA targets using the model system C. elegans.. Addition of VPA or Li+ to C. elegans affects many behaviours, one of which, defecation, is known to be regulated by IP3 inositol phosphates. Thus, both Li+ and VPA affect inositol signalling in both mammals and C. elegans. Other effects of VPA on C. elegans are: a retarded growth rate, altered levels of acetylcholine release, reduced egg laying, increased locomotion rate at low concentrations, and paralysis at high concentrations. All of these behaviours are known to be regulated by DAG, suggesting that VPA affects both IP3 and DAG signalling pathways.. To identify VPA targets we have carried out genetic screens for VPA-resistant mutants. Some of the mutants isolated from locomotion or growth assays were also resistant for the effects of VPA on defecation. Those mutations are currently being mapped. We have also tested a number of candidate mutants defective for neurotransmitter release for their response to VPA: protein kinase C mutants (pkc-1) are resistant to VPA, whereas phospholipase C beta mutants (egl-8) and diacylglycerol kinase mutants (dgk-1) are hypersensitive in the paralysis assay at high concentrations. The similar phenotype of egl-8 and dgk-1 is a surprise as they have opposite effects on DAG levels. EGL-8 produces DAG and DGK-1 phosphorylates DAG to phosphatidic acid (PA) and PA is the first step for the resynthesis of PIP2 from DAG thus both mutants might be expected to have lowered PA levels and defective resynthesis of PIP2. However, a DAG analog (PMA), which can be neither phosphorylated by DGK-1 nor be used to resynthesize PIP2, caused hypersensitivity to VPA. Thus, VPA appears to mediate at least some of its effects by altering levels of DAG.

Akiko Miyara et al. (2003) International Worm Meeting "Analysis of novel thermotaxis-defective mutants, nj21, nj34, nj35 and nj41, in C. elegans"

Akane Ohta, Masatoshi Okumura, Yoshifumi Okochi, Akiko Miyara, Ikue Mori

[International Worm Meeting, 2003]

C. elegans utilizes ambient temperature for seeking and staying around the food source in the soil. The behavioral response to temperature can be observed as thermotaxis. To elucidate mechanism of thermotaxis at cellular and molecular levels, our laboratory has been isolating and analyzing thermotaxis-defective mutants. Many mutants were isolated in our recent screen that did not rely on behavioral abnormality as the primary cue (see abstracts by Okumura et. al., IWM 2001). Among newly isolated mutants through this screen, we analyzed nj21, nj34, nj35 and nj41 mutants in this study. The nj34 mutant shows cryophilic phenotype and is partially defective in chemotaxis to NaCl. By using snip-SNPs (1), we found that nj34 maps to the region between +0.92 and +1.18 on the chromosome I. The other cryophilic mutant nj21 exhibited partially abnormal phenotypes in chemotaxis to NaCl and several odorants as well as in osmotic avoidance. The mapping results mainly based on snip-SNPs showed that nj21 maps to the region between +0.92 and +2.46 on I, where nj34 maps. Further, nj34 did not complement nj21, suggesting that these two mutations are different alleles of the same gene. No genes were previously reported to be required for thermotaxis in this region. Thus, the gene mutated in nj21 and nj34 mutants is likely to be a novel gene required for several sensory modalities. The nj35 mutant shows athermotactic phenotype on a temperature gradient, after grown at 20-degrees, although they tends to respond to cultivation temperatures, after grown at 15- and 25-degrees. A close observation of locomotion revealed that nj35 animals often lift their head for a given length of time (less than 1 second). This makes us think that nj35 may be normal in thermosensation but may show defective thermotaxis probably because of its kinetics-related abnormality on a temperature gradient. The nj41 mutant showed either cryophilic or athermotactic phenotype, after grown at 20-degrees, and cryophilic phenotype, after grown at 15- and 25-degrees. In summary, thermotactic abnormalities of these newly isolated mutants are unprecedented. Although they could simply retain gene functions, the further analysis of these mutants could give us a new important information for the mechanism of thermosensory transduction and neural plasticity. We thank Stephen Wicks, Naoki Hisamoto and Takeshi Ishihara for valuable advice on mapping with snip-SNPs. (1) Wicks et al. (2001), Nat. Genet. 28, 160.