Harris, Nathan et al. (2019) International Worm Meeting "Molecular regulators of C. elegans thermotaxis and thermosensory plasticity."

Harris, Nathan, Yu, Yanxun, Sengupta, Piali

[International Worm Meeting, 2019]

A critical feature of the nervous system is its ability to modify its function, and subsequently behavior, based on an animal's experience and context. What are the molecular pathways by which external stimuli are translated by sensory neurons into experience-dependent changes in neuronal physiology and behavior? C. elegans exhibits robust thermotaxis towards a preferred temperature when placed on a temperature gradient, and this preference depends on the animal's long-term temperature experience. The electrical and synaptic physiology of the primary sensory neuron type, AFD, is altered as a function of temperature experience, and this plasticity is necessary for correct thermotaxis. Temperature is a continuous variable, and AFD physiology and thermotaxis behavioral preference scale in relation to temperature experience. Thus, this form of plasticity operates in a graded, or 'analog' paradigm. We hypothesize that, in order to achieve this aspect of plasticity, the expression and/or function of molecular regulators of AFD physiology must also scale as a function of temperature. As a starting point to identify these molecular players, we are assessing genome-wide gene expression changes that occur in AFD after new temperature experience, using translating ribosome affinity purification (TRAP) with GFP-tagged ribosomal subunits expressed specifically in AFD, followed by RNA-Seq. We are also assessing the contribution of candidate genes known to be expressed in AFD, and whose expression appears to be differentially regulated by temperature experience from initial TRAP data, to temperature-dependent plasticity of AFD physiology. We have adopted a calcium imaging strategy that allows imaging of multiple animals simultaneously to screen candidate molecules at higher throughput. Our long-term goal is to fully describe the molecular effectors that execute long-term plasticity in AFD, as well as to identify the upstream signaling pathways that regulate the function and expression of these effectors. We hope to gain a greater understanding of regulatory principles that enable finely tuned graded plasticity, in contrast to drastic binary switches in cellular state.

Harris, Nathan et al. (2021) International Worm Meeting "Temperature-regulated gene expression changes driving plasticity in the AFD thermosensory neurons"

Harris, Nathan, Zhuang, Zihao, Calarco, John, Sengupta, Piali

[International Worm Meeting, 2021]

Animals must respond to changing external stimuli by altering their behavior and physiology in order to maintain fitness. Many of these responses rely on neuronal and neural circuit functional plasticity, which often persist long after cessation of the initial stimulus. Prolonged plasticity is typically achieved through changes in gene expression in the neurons that respond to stimuli and execute stimulus-specific behaviors. We sought to identify genes displaying expression changes in a stimulus- and cell type-specific manner, and determine both how these genes enable neuronal plasticity, and how they are engaged by external stimuli. We used the C. elegans thermosensory neuron pair AFD as a model for stimulus-dependent neuronal plasticity. C. elegans exhibits robust thermotaxis towards a preferred temperature when placed on a temperature gradient, and this preference can be set and reset as a function of the animal's long-term temperature experience. The sensory and synaptic physiology of AFD is altered as a function of temperature experience, and this plasticity is necessary for correct thermotaxis. Temperature is a continuous, rather than discrete variable, and C. elegans is able to alter its behavior and AFD physiology precisely to match temperature changes over a large dynamic range. We previously showed that temperature experience alters gene expression in AFD. However, it is largely unknown what molecular logic might enable presumably analog changes in gene expression in response to the temperature stimulus. To identify temperature-dependent gene expression changes in AFD, we used translating ribosome affinity purification (TRAP) with GFP-tagged ribosomal subunits expressed specifically in AFD, followed by RNA-Seq. We have validated the temperature-dependent expression of a number of these genes with extrachromosomal and/or endogenous reporters. We are functionally characterizing these genes with behavior and calcium imaging assays, dissecting surrounding non-coding DNA to uncover cis-regulatory features, and placing them within previously described genetic pathways controlling AFD thermosensation. Our long-term goal is to use AFD to derive general principles for how external stimuli can be transduced into gene-regulatory signals that modulate gene expression as a function of stimulus magnitude and dynamics, enabling neural plasticity that is precisely regulated as a function of variations in an animal's environment.

James B Endres et al. (2002) West Coast Worm Meeting "A Wnt gradient may guide HSN migration."

Nancy Hawkins, Gian Garriga, Shaun Cordes, James B Endres

[West Coast Worm Meeting, 2002]

The migration of neuronal cell bodies is a critical feature of neural development in all animal phyla. To investigate the molecular mechanisms of this process, we study the migrations of the hermaphrodite specific neurons (HSNs) of Caenorhabditis elegans. The HSNs are born in the tail of the comma-stage embryo and migrate anteriorly to the midbody. Later, during postembryonic development, the HSNs extend axons and synapse onto vulval muscles to control egg laying. Migrations of both the HSNs (Desai et al. 1988) and the Q neuroblasts (Harris et al. 1996) require the function of the egl-20 gene, which encodes a Wnt homolog. Wnts are secreted glycoproteins with diverse functions in many developmental processes. Desai et al. (1988) demonstrated that loss of egl-20 leads to posterior displacement (undermigration) of the HSNs, and we have observed that EGL-20 overexpression leads to anterior displacement (overmigration). Endogenous EGL-20 is expressed in the embryonic tail during the stage when the HSNs are migrating, raising the possibility that an EGL-20 gradient guides HSN migration. In contrast, EGL-20 plays a permissive role in Q cell migration by controlling mab-5 expression (Maloof et al. 1999). Supporting the possibility that an EGL-20 gradient is essential for HSN migration, global overexpression of EGL-20 from a heat-shock promoter leads to both overmigration and undermigration of the HSNs.

Hapiak, Vera et al. (2009) International Worm Meeting "Multiple monoamine receptors modulate nose touch in C. elegans."

Schafer, William, Chatzigeorgiou, Marios, Komuniecki, Richard, Wragg, Rachel, Hapiak, Vera, Harris, Gareth

[International Worm Meeting, 2009]

The ASH sensory neuron in C. elegans is polymodal, responding to both noxious (high osmolarity/volatile repellents) and mechanical (nose touch) stimuli to initiate backward locomotion. Examination of one ASH-mediated locomotory behavior, avoidance to octanol, has revealed that modulation of the ASH neural circuit is complex and involves multiple monoamines and distinct amine receptors (Wragg et al., 2007; Harris et al., 2009). In the present study, we have identified the monoamine receptors involved in another 5-HT stimulated ASH-mediated aversive response, nose touch. Like responses to octanol, multiple 5-HT receptors also have been identified in the food/5-HT sensitization of nose touch. For example, the expression of ser-5 in the ASHs appears to be essential for 5-HT dependent increases in aversive responses to nose touch. In contrast, both TA and OA inhibit nose touch and two monoamine receptors (F14D12.6 and SER-3) appear to be involved in octopaminergic inhibition. These receptors are currently being localized in the ASH locomotory circuit. Using the calcium indicator cameleon, we analyzed ASH Ca2+ responses to nose touch in ser-5 null animals. ASH Ca2+ transients were independent of exogenous 5-HT and more robust in ser-5 null animals than in wild-type or ser-4; mod-1; ser-7 ser-1 quadruple null animals that presumably only express ser-5. Since we predict that SER-5 stimulates neurotransmitter release at the ASH synapse, the increased Ca2+ signaling in the ASH soma was surprising. We are currently exploring whether SER-5 signaling has other effects on the ASH or whether SER-5 also modulates the release of other ligands, perhaps peptides, that inhibit ASH Ca2+ dynamics.

Gallotta I et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Cell Specific Knock-down of Gene Function in Targeted C. elegans Neurons"

D'Orta M, Vitale M, Castro S, Esposito G, Hilliard M A, Di Schiavi E, Gallotta I

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

An important aspect in the study of gene function is the possibility to dissect the role exerted by a gene of interest selectively in specific cells or groups of cells. In C. elegans the available approaches are time consuming and largely restricted to non-essential genes for which mutants are available. We have developed a simple reverse genetics approach to reduce the function of specific genes in chosen C. elegans neurons (Esposito et al., 2007). By expressing sense and antisense RNA corresponding to a gene of interest under cell-specific promoters, we obtain an efficient, heritable and cell autonomous knock-down of the targeted gene function in the specific neurons. This strategy has now been used by several other labs, with a variety of genes and on different neurons (Harris et al. 2009; Yamada et al. 2009; Jose et al. 2009; Ezak et al. 2010). To optimize the efficiency of this approach we have tested the influence of genetic backgrounds that affect RNA interference. We show that in a rrf-3(pk1426) background silencing is more efficient. Although this result indicates the involvement of RNAi pathways in the silencing mechanism we have shown that there is no spreading of the silencing effect at least among amphidial chemosensory neurons and within the motorneurons of the ventral cord. We are now targeting chemosensory and mechanosensory neurons and two classes of motorneurons (GABAergic and HSN) using a variety of different promoters. We are testing the effect of silencing several essential genes on the development, morphology and survival of these neurons as well as on the behavior that they control.

Chung, Kevin et al. (2015) International Worm Meeting "The neuronal basis of bacterial food odor preference."

Kan, Emily, Glater, Elizabeth, Haynes, Lillian, Ota, Ryan, Chambers, Melissa, Chung, Kevin

[International Worm Meeting, 2015]

Caenorhabditis elegans uses chemosensation to distinguish among various species of bacteria, their major food source 1-3. Although the neurons required for the detection of specific food-odors have been well-defined, less is known about the sensory circuits underlying the discrimination among the mixtures of odors released by different kinds of bacteria. We are examining the neural machinery underlying bacterial preference among a diverse set of bacterial species. Specifically, we are testing the food preferences of C. elegans for bacteria found in their natural habitats (kindly provided by Marie-Anne Felix, Institute of Biology of Ecole Normale Superieure, Paris, France). We have found that C. elegans prefers the odors of most species tested over E. coli. We have identified that the olfactory neuron AWC is involved in many preferences. We also find that some bacterial choices involve multiple sensory neurons with opposing roles. For example, in one choice in which wild-type animals prefer Providencia sp. (JUb39) over E. coli, the AWC neuron appears to be involved in increased preference for Providencia and the AWA neuron in increased preference for E. coli. In the future we will extend our analysis to additional bacterial species to determine the diversity of the underlying neuronal mechanisms.1. Harris, G. et al. Dissecting the Signaling Mechanisms Underlying Recognition and Preference of Food Odors. J. Neurosci. 34, 9389-9403 (2014).2. Ha, H. et al. Functional Organization of a Neural Network for Aversive Olfactory Learning in Caenorhabditis elegans. Neuron 68, 1173-1186 (2010).3. Shtonda, B. B. & Avery, L. Dietary choice behavior in Caenorhabditis elegans. J. Exp. Biol. 209, 89-102 (2006).

Harris, Gareth P. et al. (2009) International Worm Meeting "Neuropeptides are essential for the serotonergic stimulation of aversive responses mediated by the ASH sensory neurons."

Summers, Philip, Hapiak, Vera, Komuniecki, Richard, Wragg, Rachel, Korchnak, Amanda, Harris, Gareth P.

[International Worm Meeting, 2009]

Serotonin modulates many key behaviors in C. elegans, including the stimulation of aversive responses to dilute octanol mediated by the ASH sensory neurons (Harris et al., 2009, J. Neuroscience 29, 1446-1456). The serotonergic stimulation of ASH-mediated aversive responses requires the expression of SER-5 in the ASHs, but the site of SER-5 action is unclear. For example, SER-5 signaling may increase the release of glutamate and/or neuropeptides from the the ASHs. Indeed, the ASHs express multiple peptide encoding genes, including nlp-3 and nlp-15, and the peptides encoded by these genes have multiple effects on ASH signaling. For example, nlp-3 null animals exhibit wild-type basal responses to dilute octanol, but do not increase aversive responses in the presence of food or 5-HT. In contrast, nlp-15 null animals exhibit elevated basal responses to dilute octanol in the absence of either food or 5-HT and these elevated basal responses are not inhibited by octopamine. As predicted, animals overexpressing nlp-15 respond only weakly to dilute octanol and exhibit dramatically reduced basal responses. Interestingly, in these nlp-15 overexpressors, aversive responses can be stimulated to near wild-type levels by food or 5-HT. Together, these results suggest that nlp-15 inhibits ASH signaling by a mechanism that may not directly involve NT release from the ASHs. These observations have been confirmed by the ASH rescue and RNAi knockdown of nlp-15 and nlp-3. In addition, we have identified a number of other genes encoding peptides and peptide receptors that are also involved in modulating various aspects of ASH mediated signaling, highlighting the complexity of peptidergic modulation. These studies are continuing with the goal of identifying the receptors and downstream signaling pathways mediating the differential effects of nlp-3 and nlp-15 on signaling in the ASH sensory neurons.

Clark SG et al. (1997) International C. elegans Meeting "MIG-1 ENCODES A FRIZZLED-RELATED RECEPTOR"

Clark SG, Bargmann CI

[International C. elegans Meeting, 1997]

mig-1 specifies cell fates and guides cell migrations along the anteroposterior body axis of C. elegans. In wild type animals, the descendants of the neuroblast QL migrate posteriorly, whereas the descendants of the neuroblast QR migrate anteriorly. In mig-1 mutants, the QL descendants migrate anteriorly like the QR descendants, suggesting that mig-1 determines the fates of the QL descendants (1, 2). mig-1 is also required for the normal migration and positioning of the mesoblast M and the HSNs (2, 3). We determined that mig-1 and cfz-1, which encodes a frizzled-related transmembrane protein (4), are the same gene. Like other frizzled-related genes, mig-1/cfz-1 encodes a protein with an amino-terminal signal sequence, a conserved cysteine-rich extracellular domain and seven membrane-spanning domains. frizzled-related proteins have been shown to act as wingless or wnt receptors, suggesting that MIG-1 is also a wnt receptor. The migration defects conferred by mig-1 mutations vary in severity and penetrance. The canonical mig-1 allele, e1787, is a null mutation by molecular criteria, suggesting that multiple guidance processes influence these migrations. To examine the expression pattern of mig-1, we constructed a mig-1::gfp translational fusion. mig-1::gfp is membrane localized and first expressed in hypodermal cells during embryogenesis. We also observed expression in some neurons (HSN, QR, QL, SIA, SIB), body wall muscle and M. We are currently investigating whether mig-1 acts within the affected migratory cells or within the surrounding tissues that might influence their migrations or fates. 1. Hedgecock, E. et al., Development 100: 365-382 (1987). 2. Harris, J. et al., Development 122: 3117-3131 (1996). 3. Desai, C. et al., Nature 336: 638-646 (1988). 4. Wang, Y. et al., J. Biol. Chem. 271: 4468-4476 (1996).

Burr AHJ (1995) International C. elegans Meeting "LOCOMOTORY MECHANICS OF ASCARIS"

Burr AHJ

[International C. elegans Meeting, 1995]

For freshly collected Ascaris a loop of silk thread tightened about the anterior elicits a continuous anteriorly-directed wave. In a 16 mm wide tube, the body waves wedge the animal between the sides and the animal progresses forward. Similar behavior, elicited by the flow of intestinal contents against the head, may be responsible for maintaining the worm's position in the intestine. The posterior two thirds where the ovary fills the body cavity does not bend appreciably. The three anteriorly directed waves initiate in the midbody region behind the ovary and propulsion is accomplished by the more active and muscular anterior third of the body. The worm progresses anteriorly because the body in contact with the wall of the tube rolls along the wall as the bends propagate anteriorly. The posterior half is dragged along. This mode of locomotion is not known in any other animal - snakes in tubes utilize another method of crawling. The bending motion will be analysed with reference to the boundaries of the first three repeating motor segments (the location of DE2 commissures were marked to be visible in the video recordings). The body shortens when the internal fluid volume is reduced by removing pseudocoelomic fluid. Length-tension curves indicate that the longitudinal body wall muscle resets to a shorter operating range, possibly to restore internal pressure. Bending motion and locomotion was observed in volume-reduced worms. Bending was observed even when the pseudocoelomic fluid was continuously vented through a cannula. This controverts the hypothesis that pseudocoelomic hydrostatic pressure provides the restoring force to bending during locomotion (Harris and Crofton, 1957). It is possible that an opposing hydrostatic pressure can develop in the considerable cytoplasmic volume of the muscle bellies.

Christopher Hopkins et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Probing UNC-18 Interactions to SNARE Proteins: Pull-downs, Phosphorylation Dependence, and Affinity Measurements"

Christopher Hopkins, Erik Jorgensen

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Synaptic transmission requires the SNARE complex proteins syntaxin, SNAP-25, and synaptobrevin. UNC-18 is also required for neurotransmitter release but its role remains controversial. Affinity experiments with recombinant proteins demonstrate UNC-18 retains a high affinity interaction to syntaxin but does not interact with SNAP-25 or synaptobrevin. UNC-18 may be a negative regulator of fusion by binding syntaxin and inhibiting SNARE complex formation. Alternatively, in yeast, the UNC-18 homolog (Sec1p) appears to be a facilitator of SNARE complex formation because interaction with its cognate syntaxin homolog (SSO1p) occurs only when a SNARE complex is formed. To determine if UNC-18 functions before or after SNARE complex formation, UNC-18 protein complexes were purified from UNC-18::GFP rescued C. elegans via anti-GFP affinity chromatography. As expected, western blots demonstrate that syntaxin co-immunopurifies with the UNC-18::GFP. To demonstrate specificity, a point mutation (R39C) of UNC-18 was found not to co-purify syntaxin. Probing with anti-SNAP-25 or anti-synaptobrevin failed to yield a strong signal, thus the isolated UNC-18 complex is predominantly an interaction to syntaxin and not to a SNARE complex. Unc-18 is a phosphoprotein with strong phosphorylation consensus sequences for phosphorylation by PKC, cdk5, and CAMKII. The inclusion of phosphatase inhibitors in lysis conditions leads to a dramatic reduction in syntaxin co-purification, which suggest phosphorylation mediates interaction of UNC-18 and syntaxin. With phosphatase inhibitors, an increase in SNAP-25 co-purification was observed (synaptobrevin not tested). Therefore phosphorylation of UNC-18 and its binding partners appears to be critical to their interaction affinity. To directly measure UNC-18 affinity to its binding partners a chip-based TIRF method is being developed (in collaboration with Joel Harris at the U of Utah) to directly measure on rate and off rates at the single molecule level.