Yu, A. et al. (2017) International Worm Meeting "Non-associative learning as a behavioral strategy to promote dispersal."

Rankin, C., Yu, A., Ardiel, E.

[International Worm Meeting, 2017]

Habituation is a form of non-associative learning defined as decremented likelihood and/or magnitude in response to repeatedly presented stimuli. Habituation is thought to help animal ignore recurring innocuous events and free up its neural resources to attend to other novel or salient stimuli in the environment. C. elegans is known to habituate to harmless stimuli such as non-localized tap (Rankin et al., 1990). Recently, we have reported that worms also displayed behavioral characteristics of habituation when a pair of nociceptor neurons, ASH, were repeatedly stimulated with optogenetics (Ardiel et al., 2016). Because ASH detects natural aversive stimuli that are harmful and potentially lethal to worms, it seems puzzling that animals habituate to these noxious stimuli. Our analysis used a non-response-centric approach and found that habituation of ASH-mediated reversal responses are accompanied by the simultaneous sensitization of forward locomotion (Ardiel et al., 2017, unpublished). This behavioral plasticity was found to be mediated by the Pigment Dispersal Factor (PDF) signaling pathway. These coordinated changes in different behavioral components form an optimal behavioral strategy to guide animals to spend less time responding to the recurrent noxious stimuli and more time moving away from them. In this present study, we investigated whether tap habituation was associated with changes in other behavioral components. We observed similar patterns of behavioral plasticity in tap habituation that is also mediate by PDF signaling. Our findings suggest that different forms of non-associative learning participate in a suite of coordinated behavioral changes that together promote a shift in behavioral strategy. Our study also highlights the needs for more detailed behavioral analyses.

Yu, A. et al. (2019) International Worm Meeting "Neuropeptides differentially mediate facilitatory forms of non-associative learning."

Chew, Y. L., Schafer, W., Ardiel, E., Rankin, C., Yu, A.

[International Worm Meeting, 2019]

Sensitization and dishabituation are two forms of facilitatory non-associative learning produced by novel or noxious stimuli, that both increase the likelihood and/or magnitude of a response. Although these two forms of non-associative learning have similar effects on behaviour, previous research suggests that they emerge at different developmental stages and have non-identical electrophysiological profiles. Research into the cellular and molecular mechanisms of sensitization and dishabituation can help to further differentiate the two forms of learning, and provide insight into the relationship between different forms of non-associative learning. Our studies found that neuropeptides differentially mediate sensitization and dishabituation. Using our high-throughput Multi-Worm Tracker in combination with optogenetics, we established paradigms for cross-modal sensitization and dishabituation. We identified a FLP-20/FRPR-3 peptidergic signalling pathway that specifically mediates tap-induced ASH response sensitization but not dishabituation. The PDF neuropeptide system also mediates sensitization and dishabituation of the ASH response, with the possible involvement of a novel receptor. Taken together, our data suggest that a number of neuropeptide systems underlie different forms of non-associative learning. Unravelling the cellular and molecular mechanisms of non-associative learning will further our understanding of behavioural plasticity.

T Yu et al. (1999) International C. elegans Meeting "Mechanisms of axon pathfinding by SAX-3"

K Prehoda, CI Bargmann, T Yu, J Hao, W Lim, J Zallen, M Tessier-Lavigne, D Lee

[International C. elegans Meeting, 1999]

We are studying the mechanisms by which the axon guidance receptor sax-3 directs axonal outgrowth. sax-3 mutants exhibit a variety of axon phenotypes, including anterior misrouting of axons in the nerve ring, abnormal midline crossing in the ventral cord, and defects in ventrally-directed guidance (e.g. AVM, HSN). sax-3 encodes a transmembrane protein with five immunoglobulin and three fibronectin type III repeats, and has close homologs in flies ( roundabout ) and vertebrates (rat and human Robo1 , Robo2 ). Expression of sax-3 in AVM under the mec-7 promoter rescues its ventral defect, arguing that sax-3 acts as a receptor. What is the signal to which sax-3 responds? One candidate is slit , a secreted molecule first identified in a screen for midline CNS mutants in flies. Experiments with a worm homolog of slit demonstrate that high levels of slit expression are found in regions avoided by sax-3 -dependent axons. In particular, slit is expressed strongly in dorsal, but not ventral, body wall muscle, and in epidermal cells that lie at the anterior boundary of the nerve ring. This expression pattern suggests a model in which sax-3 is a repulsive receptor that directs axons away from sources of slit . In support of this model, misexpression of slit in ventral muscles is sufficient to disrupt AVM ventral growth, an effect which is dependent upon the presence of sax-3 . How does sax-3 elicit changes in growth cone behavior? Binding studies show that a conserved motif in the cytoplasmic domain of sax-3 interacts with enabled, a protein implicated in actin assembly, Listeria motility and cell protrusion. In support of a role of enabled in sax-3 guidance, mutations in unc-34 , a worm homolog of enabled, show strong genetic interactions with sax-3 , in which weak unc-34 alleles dominantly enhance axon defects of a weak sax-3 allele. We have found that a second conserved motif in SAX-3 binds to a C. elegans homolog of dreadlocks , a SH2/SH3 adapter molecule first identified in flies in a screen for axon guidance mutants in the visual system. Currently we are focusing on obtaining more functional evidence for role of unc-34 / ena and dock in sax-3 guidance. These studies may provide a link between guidance events at the membrane and known modulators of the cytoskeleton.

Yu, Alex et al. (2021) International Worm Meeting "Charactering the roles of neuropeptides in non-associative learning"

Rankin, Catharine, Yu, Alex

[International Worm Meeting, 2021]

Non-associative forms of learning (habituation, dishabituation and sensitization) are the simplest forms of learning and are often thought as "cognitive building blocks" for higher learning. To better understand non-associative forms of plasticity and the cellular and molecular mechanisms, our work focuses on neuromodulatory signalling pathways underlying these forms of plasticity. First, we found that egl-3 and egl-21 mutants deficient of neuropeptides showed learning deficits in three forms of non-associative learning, habituation, dishabituation, and sensitization,, suggesting neuropeptides play a key role in non-associative learning. Further, we found that FLP-20/FRPR-3 signalling mediates sensitization but not dishabituation of the same response, and that the PDF neuropeptides differentially affect sensitization of response duration and response speed. These data suggest different forms of non-associative plasticity are mediated by different cellular and molecular components. We are in the process of testing mutations in a range of neuropeptide genes to examine how they influence non-associative learning. This project will generate data to better understand how neuropeptide signalling mediates different forms of non-associative plasticity.

Yu L et al. (2000) East Coast Worm Meeting "TGF-beta Signaling and Egg Laying."

Tokarz R, Yu L, Savage C

[East Coast Worm Meeting, 2000]

A TGF-beta -related signaling pathway regulates dauer larval development and egg laying in C. elegans. Mutations in daf-7 ligand, daf-1 type I receptor, daf-4 type II receptor, and daf-8 and daf-14 Smads result in Dauer-constitutive and Egg-laying defective animals. These mutations are suppressed for both defects by mutations in daf-3 Smad. We are interested in the role of this pathway in egg laying. Two other genes that affect egg laying, egl-4 and egl-32, are also implicated in this pathway by their suppression by daf-3. In addition, egl-4 mutants have defects in chemotaxis (S.A. Daniels and P. Sengupta, pers. comm.). Because egg laying and dauer formation are influenced by environmental cues, it is possible that all of these phenotypes have a common cause in defective chemosensory neuron function. We first determined whether other egg-laying defective mutants could be suppressed by daf-3, but found no additional genes of this class. We then undertook a molecular analysis of egl-32. We narrowed down the egl-32 interval using chromosomal deficiencies to the region between unc-29 and mec-8 on LGI. One cosmid from this interval, C26G6, which overlaps the sequenced cosmid T08G11, rescued the egl-32 phenotype. Among several subclones of this cosmid, one containing two predicted ORFs T08G11.2 and T08G11.3, but not one containing only T08G11.3, also rescued egl-32. Therefore, egl-32 likely corresponds to predicted ORF T08G11.2. We further tested this hypothesis by RNAi, and found that injection of L4 animals by dsRNA from this gene results in the Egl defect. Injection of dsRNA into adults gave no phenotypes in the offspring. The ability of late dsRNA injection to phenocopy the egg-laying defect is consistent with a defect in chemosensory function rather than a developmental defect. The predicted ORF encodes a small (282 aa) protein with a putative SH2 domain. How such a protein fits in with TGF-beta signaling is an intriguing problem.

Yu TW et al. (1997) International C. elegans Meeting "Genetic Analysis of Axonal Guidance Cues"

Bargmann CI, Zallen JA, Yu TW

[International C. elegans Meeting, 1997]

In the developing C. elegans nervous system, neurons extend processes through a complex extracellular environment to establish a remarkably stereotyped network of connections. We are interested in understanding the mechanisms that direct the proper formation of these connections. Recently, a genetic screen was conducted for mutants defective in the formation of the C. elegans nerve ring (abstract, J. Zallen and C. Bargmann). One gene identified in this screen, sax-3 (sensory axon defects), encodes a predicted transmembrane protein with immunoglobulin-like domains and fibronectin type-3 repeats. sax-3 worms exhibit multiple defects in cell and axon migrations along both longitudinal and circumferential axes. The structural motifs present in SAX-3 are reminiscent of a number of recently characterized molecules that affect axon guidance and cell migration. Little is known about the signaling mechanisms by which these molecules exert their effects. Accordingly, we are interested in identifying other genes that interact with sax-3. To this end, we are using a temperature-sensitive sax-3 allele (ky200) as the basis for conducting sensitized screens for enhancers and suppressors of sax-3. The identification of sax-3 ligands and downstream signaling elements will extend our understanding of the relationship between events at the cell surface and cell and axon guidance.

Yu, C. et al. (2013) International Worm Meeting "A high-throughput genetic screen for lethargus mutants."

Yu, C., Fang-Yen, C., Churgin, M., Raizen, D.

[International Worm Meeting, 2013]

Lethargus is a C. elegans sleep-like quiescent state which occurs during the four larval molt transitions and is characterized by cessation of feeding and locomotion. Current methods for assaying quiescence are limited to <10 animals, making large-scale genetic screening impractical. We have developed a high-throughput quiescence assay capable of simultaneously imaging activity in hundreds of uniquely identified animals during development. Our assay is based on the WorMotel, a microfabricated polydimethylsiloxane (PDMS) device containing an array of 240 wells optimized for C. elegans cultivation and imaging, each containing one worm on standard agar media. The WorMotels are integrated in a standard 384-well microplate format for compatibility with multichannel pipettes, microplate handling equipment, and the COPAS worm sorter. In our experiments, we transfer synchronized populations of L3 larva into wells containing DA837-seeded NGM agar and perform time-lapse imaging of the array with a single camera over a 24 h period. We use custom MATLAB programs to quantify the activity and total locomotory quiescence of individual worms. Using our quiescence assay on N2 animals we find an L3 lethargus duration of 1.4 plus or minus 0.4 h and L4 lethargus duration of 2.0 plus or minus 0.4 h, consistent with published data based on assays in standard agar plates. We validate our assay on mutants with known lethargus phenotypes including egl-4 (increased quiescence), kin-2 (decreased quiescence), and other wild-type strains of C. elegans. We describe efforts toward identifying novel mutants with reduced movement quiescence during lethargus in EMS-mutagenized animals. Our method will also be useful for conducting high-throughput assays for other behavioral and developmental phenotypes.

Lu, Yu et al. (2009) International Worm Meeting "Centrosome elimination during C. elegans development."

Roy, Richard, Lu, Yu

[International Worm Meeting, 2009]

Many sexually reproducing organisms must eliminate a pair of centrioles from their gametes prior to the first zygotic division to avoid the formation of a multipolar spindle. In the C. elegans germ line, centrosomes are eliminated just prior to oocyte specification and this process requires the activity of the p21/p27-like CDK inhibitor protein cki-2, which may function by antagonizing cyclin E/CDK-2. This suggests that changes in CDK activity are required for this process and altering CDK activities may affect proper centrosome elimination. We therefore decided to examine whether CDKs exert these effects through proteins important for centrosome duplication. In addition to oocyte-specific elimination of the centrosome, we found that centrosomes are eliminated from cells undergoing endocycles. The intestinal and hypodermal cells undergo endoreduplication during postembryonic development. Using a SPD-2::GFP reporter to detect the presence of centrosomes in these tissues, we observed that the centrosomes were gradually lost from intestinal nuclei after the L1 stage, while they disappeared from the hypodermal cells at the L2 stage. Using an anti-SPD-2 antibody, we further confirmed that the intestinal cells actively eliminate their centrosomes at the onset of endoreduplication. We are therefore using a candidate gene approach in the intestine while monitoring SPD-2::GFP to address the molecular mechanism underlying this process. Interestingly, SPD-2 possesses multiple canonical CDK phosphorylation sites and we mutated the highly predicted sites to generate SPD-2 variants. Subsequently we examined them in various developmental contexts where centrosome elimination occurs. When we introduced the non-phosphorylable SPD-2 variant we noted extra centrosomes in the intestine, which were visible after the L2 stage, when the centrosomes are normally eliminated, suggesting that the proper phosphorylation of SPD-2 may contribute to timely centrosome elimination in the intestine. We are currently testing phosphomimetic SPD-2 for its effects. Considering that continued CDK-mediated phosphorylation results in centrosome persistence in the germ line, our observation in the intestine suggests that perhaps a second means of triggering centrosome elimination may function in the endocycling gut that is distinct from that during oocyte specification, but may nevertheless require CDK-mediated phosphorylation.

Tim Yu et al. (2001) International C. elegans Meeting "Roles of unc-34 and unc-40 in repellent guidance by sax-3"

Wendell Lim, Cori Bargmann, Tim Yu, Marc Tessier-Lavigne, Joe Hao

[International C. elegans Meeting, 2001]

Attractive and repulsive guidance receptors play key roles in the development of the nervous system by directing growing axons to their proper targets. The signaling mechanisms by which these receptors communicate with the growth cone cytoskeleton are not well understood. We are studying mechanisms of axon repulsion mediated by sax-3 , a member of the Robo family of guidance receptors. sax-3 mutants have defects in anterior and posterior cell migrations, in the formation of the nerve ring in the head, and in ventral axon guidance in the body. The slt-1 gene, a homolog of Drosophila and vertebrate Slit, encodes one probable ligand for sax-3 in several of these guidance decisions. Members of the Enabled/VASP family of proteins have been shown to be important modulators of actin dynamics and are potential downstream effectors of axon guidance receptors. We find that the C. elegans Enabled protein UNC-34 is a mediator of SAX-3 signaling in repulsive guidance from SLT-1. Genetic interactions with gain and loss of function mutations in the sax-3 pathway demonstrate that unc-34 works together with sax-3 in several guidance decisions. UNC-34 associates with the SAX-3 cytoplasmic domain in vitro , suggesting that these interactions are direct. These genetic and biochemical interactions are conserved across species, since Robo and Enabled also act together in Drosophila midline guidance (Bashaw et al, 2000). These results support a direct role for UNC-34 in repellent guidance. Unexpectedly, SAX-3 function is also potentiated by an UNC-6/Netrin-independent function of the Netrin receptor UNC-40. Genetic interactions between unc-40 and sax-3 are similar to interactions between unc-34 and sax-3 , and point to a role for UNC-40 in SAX-3 guidance. Such a role is further supported by the finding that UNC-40 and SAX-3 cytoplasmic domains directly interact. The interaction between UNC-40 and SAX-3 is also observed with their vertebrate counterparts, DCC and Robo, where it is implicated in a different guidance decision (Stein et al, 2001). Our results suggest that SAX-3 and UNC-40 may interact directly in guidance complexes that contribute to repulsive guidance. A combinatorial logic thus dictates alternative functions for UNC-40/DCC, which can act in attraction to UNC-6, repulsion from UNC-6 (with UNC-5), or repulsion from Slit (with SAX-3).

Xiang Yu et al. (2004) East Coast Worm Meeting "Candidate EGO-1 interactors that function in germline development."

Bill Kelly, Xiang Yu, Tom Ratliff, Jamie Wasilenko, Eleanor Maine, Valarie Vought

[East Coast Worm Meeting, 2004]

ego-1 was identified in a genetic screen for e nhancers of g lp- 1 (Qiao et al .1995). Later studies showed that ego-1 mutants have defects in germline proliferation, meiotic progression, gametogenesis and RNA interference (RNAi) (Smardon et al . 2000). EGO-1 belongs to the RNA-dependent RNA Polymerase (RdRP) family. RdRP family members have been shown to function in RNA silencing in many organisms, although their specific role is unclear. Our working model is that the ego-1 developmental defects result from defects in RNA metabolism, perhaps specifically from the failure to produce or amplify small, non-coding RNA important for various cellular processes. Two approaches that we have taken to understanding the role of EGO-1 in germline development are to screen for interaction partners using the yeast two-hybrid system and to investigate chromatin structure in ego-1 mutant germ lines. EGO-1 is predicted to be a 1632 amino acid (aa) protein with a conserved RdRP domain (813 aa) located between N-terminal (491 aa) and C-terminal (328 aa) domains. We used both N-terminal and C-terminal domains as "bait" in two-hybrid screens. We then tested candidate interactors by RNAi to identify those that function in the germ line, and particularly those whose germline function might be related to ego-1 . We identified three genes that bind the EGO-1 N-terminal domain and have severe proliferation defects in RNAi injection experiments. We then used tempered RNAi to produce milder proliferation defects, and checked for genetic interactions with glp-1 . We find that R08C7.12 RNAi causes a meiotic progression defect similar to ego-1 mutants, but does not enhance glp-1(ts) . In contrast, ZC518.2 RNAi causes a masculinized germ line (a Mog defect) in wildtype animals and enhances glp-1(ts) . Finally, the Y54F10BM.2 RNAi proliferation defect is more severe in a glp-1(ts) background than in a wildtype background, suggesting that it is enhanced by glp-1(ts) . We are now extending these RNAi studies to include other relevant genes, and are collaborating with the Conradt lab (Dartmouth College) to recover deletion alleles of all three genes. To complement these studies, we are using a directed yeast two-hybrid approach to better define the physical interactions between EGO-1 and these three proteins. Studies in S. pombe have shown that chromatin silencing modifications depend, at least in part, on components of the RNAi machinery, including the pombe RdRP, RdP1 (Volpe et al. , 2002). For example, accumulation of a silencing modification on centromeres, methylation of histone H3 on the lysine 9 residue (H3meK9), is defective in RdP1 mutants. In the C. elegans germ line, H3meK9 silencing modifications accumulate on the X chromosome during male meiosis and on other unpaired DNA (Bean et al ., 2004). We are investigating whether EGO-1 function is important for this aspect of germline chromatin assembly. Preliminary data suggest that silencing modifications on the male X are dependent on EGO-1 function. Bean et al . (2004) Nature Genetics 36, 100-105 Qiao et al. (1995) Genetics 141, 551-569 Smardon et al . (2000) Current Biology 10, 169-178 Volpe et al . (2002) Science 297, 1833-1837.