Selected Publications

Most eukaryotic cells express small regulatory RNAs. The purpose of one class, the somatic endogenous siRNAs (endo-siRNAs), remains unclear. Here, we show that the endo-siRNA pathway promotes odor adaptation in C. elegans AWC olfactory neurons. In adaptation, the nuclear Argonaute NRDE-3, which acts in AWC, is loaded with siRNAs targeting odr-1, a gene whose downregulation is required for adaptation. Concomitant with increased odr-1 siRNA in AWC, we observe increased binding of the HP1 homolog HPL-2 at the odr-1 locus in AWC and reduced odr-1 mRNA in adapted animals. Phosphorylation of HPL-2, an in vitro substrate of the EGL-4 kinase that promotes adaption, is necessary and sufficient for behavioral adaptation. Thus, environmental stimulation amplifies an endo-siRNA negative feedback loop to dynamically repress cognate gene expression and shape behavior. This class of siRNA may act broadly as a rheostat allowing prolonged stimulation to dampen gene expression and promote cellular memory formation

Most eukaryotic cells express small regulatory RNAs. The purpose of one class, the somatic endogenous siRNAs (endo-siRNAs), remains unclear. Here, we show that the endo-siRNA pathway promotes odor adaptation in C. elegans AWC olfactory neurons. In adaptation, the nuclear Argonaute NRDE-3, which acts in AWC, is loaded with siRNAs targeting odr-1, a gene whose downregulation is required for adaptation. Concomitant with increased odr-1 siRNA in AWC, we observe increased binding of the HP1 homolog HPL-2 at the odr-1 locus in AWC and reduced odr-1 mRNA in adapted animals. Phosphorylation of HPL-2, an in vitro substrate of the EGL-4 kinase that promotes adaption, is necessary and sufficient for behavioral adaptation. Thus, environmental stimulation amplifies an endo-siRNA negative feedback loop to dynamically repress cognate gene expression and shape behavior. This class of siRNA may act broadly as a rheostat allowing prolonged stimulation to dampen gene expression and promote cellular memory formation

Genetics. 2019, July 22; 212(3):1010-1022. doi: 10.1534/genetics.119.302392

Sarah Woldemariam, Jatin Nagpal, Tyler Hill, Joy Li, Martin W. Schneider, Raakhee Shankar, Mary Futey, Aruna Varshney, Nebat Ali, Jordan Mitchell, Kristine Andersen, Benjamin Barsi-Rhyne, Alan Tran, Wagner Steuer Costa, Michelle C. Krzyzanowski, Yanxun V. Yu, Chantal Brueggemann, O. Scott Hamilton, Denise M. Ferkey, Miri VanHoven, Piali Sengupta, Alexander Gottschalk and Noelle L'Etoile

cGMP plays a role in sensory signaling and plasticity by regulating ion channels, phosphodiesterases and kinases. Studies that primarily used genetic and biochemical tools suggest that cGMP is spatiotemporally regulated in multiple sensory modalities. FRET- and GFP-based cGMP sensors were developed to visualize cGMP in primary cell culture and Caenorhabditis elegans to corroborate these findings. While a FRET-based sensor has been used in an intact animal to visualize cGMP, the requirement of a multiple emission system limits its ability to be used on its own as well as with other fluorophores. Here, we demonstrate that a C. elegans codon-optimized version of the cpEGFP-based cGMP sensor FlincG3 can be used to visualize rapidly changing cGMP levels in living, behaving C. elegans. We coexpressed FlincG3 with the blue light-activated guanylyl cyclases BeCyclOp and bPGC in body wall muscles and found that the rate of change in FlincG3 fluorescence correlated with the rate of cGMP production by each cyclase. Furthermore, we show that FlincG3 responds to cultivation temperature, NaCl concentration changes and sodium dodecyl sulfate in the sensory neurons AFD, ASEL/R and PHB, respectively. Intriguingly, FlincG3 fluorescence in ASEL and ASER decreased in response to a NaCl concentration upstep and downstep, respectively, which is opposite in sign to the coexpressed calcium sensor jRGECO1a and previously published calcium recordings. These results illustrate that FlincG3 can be used to report rapidly changing cGMP levels in an intact animal and that the reporter can potentially reveal unexpected spatiotemporal landscapes of cGMP in response to stimuli.

Elife. 2016 Jul 6;5. pii: e14000. doi: 10.7554/eLife.14000

Christine E ChoChantal BrueggemannNoelle D L'EtoileCornelia I Bargmann

Sensory experience modifies behavior through both associative and non-associative learning. In Caenorhabditis elegans, pairing odor with food deprivation results in aversive olfactory learning, and pairing odor with food results in appetitive learning. Aversive learning requires nuclear translocation of the cGMP-dependent protein kinase EGL-4 in AWC olfactory neurons and an insulin signal from AIA interneurons. Here we show that the activity of neurons including AIA is acutely required during aversive, but not appetitive, learning. The AIA circuit and AGE-1, an insulin-regulated PI3 kinase, signal to AWC to drive nuclear enrichment of EGL-4 during conditioning. Odor exposure shifts the AWC dynamic range to higher odor concentrations regardless of food pairing or the AIA circuit, whereas AWC coupling to motor circuits is oppositely regulated by aversive and appetitive learning. These results suggest that non-associative sensory adaptation in AWC encodes odor history, while associative behavioral preference is encoded by altered AWC synaptic activity.

PLoS Genet. 2009 Dec;5(12):e1000761. doi: 10.1371/journal.pgen.1000761. Epub 2009 Dec 11

Damien M. O'Halloran, Svetlana Altshuler-Keylin, Jin I. Lee, Noelle D. L'Etoile

While most sensory neurons will adapt to prolonged stimulation by down-regulating their responsiveness to the signal, it is not clear which events initiate long-lasting sensory adaptation. Likewise, we are just beginning to understand how the physiology of the adapted cell is altered. Caenorhabditis elegans is inherently attracted to specific odors that are sensed by the paired AWC olfactory sensory neurons. The attraction diminishes if the animal experiences these odors for a prolonged period of time in the absence of food. The AWC neuron responds acutely to odor-exposure by closing calcium channels. While odortaxis requires a Galpha subunit protein, cGMP-gated channels, and guanylyl cyclases, adaptation to prolonged odor exposure requires nuclear entry of the cGMP-dependent protein kinase, EGL-4. We asked which candidate members of the olfactory signal transduction pathway promote nuclear entry of EGL-4 and which molecules might induce long-term adaptation downstream of EGL-4 nuclear entry. We found that initiation of long-term adaptation, as assessed by nuclear entry of EGL-4, is dependent on G-protein mediated signaling but is independent of fluxes in calcium levels. We show that long-term adaptation requires polyunsaturated fatty acids (PUFAs) that may act on the transient receptor potential (TRP) channel type V OSM-9 downstream of EGL-4 nuclear entry. We also present evidence that high diacylglycerol (DAG) levels block long-term adaptation without affecting EGL-4 nuclear entry. Our analysis provides a model for the process of long-term adaptation that occurs within the AWC neuron of C. elegans: G-protein signaling initiates long-lasting olfactory adaptation by promoting the nuclear entry of EGL-4, and once EGL-4 has entered the nucleus, processes such as PUFA activation of the TRP channel OSM-9 may dampen the output of the AWC neuron.

Proc Natl Acad Sci U S A. 2010 Mar 30;107(13):6016-21. doi: 10.1073/pnas.1000866107. Epub 2010 Mar 10

Jin I. LeeDamien M. O'HalloranJeffery Eastham-AndersonBi-Tzen JuangJulia A. KayeO. Scott HamiltonBluma LeschAndrei Goga, and Noelle D. L'Etoile

To navigate a complex and changing environment, an animal's sensory neurons must continually adapt to persistent cues while remaining responsive to novel stimuli. Long-term exposure to an inherently attractive odor causes Caenorhabditis elegans to ignore that odor, a process termed odor adaptation. Odor adaptation is likely to begin within the sensory neuron, because it requires factors that act within these cells at the time of odor exposure. The process by which an olfactory sensory neuron makes a decisive shift over time from a receptive state to a lasting unresponsive one remains obscure. In C. elegans, adaptation to odors sensed by the AWC pair of olfactory neurons requires the cGMP-dependent protein kinase EGL-4. Using a fully functional, GFP-tagged EGL-4, we show here that prolonged odor exposure sends EGL-4 into the nucleus of the stimulated AWC neuron. This odor-induced nuclear translocation correlates temporally with the stable dampening of chemotaxis that is indicative of long-term adaptation. Long-term adaptation requires cGMP binding residues as well as an active EGL-4 kinase. We show here that EGL-4 nuclear accumulation is both necessary and sufficient to induce long-lasting odor adaptation. After it is in the AWC nucleus, EGL-4 decreases the animal's responsiveness to AWC-sensed odors by acting downstream of the primary sensory transduction. Thus, the EGL-4 protein kinase acts as a sensor that integrates odor signaling over time, and its nuclear translocation is an instructive switch that allows the animal to ignore persistent odors.

Neuron. 2009 Jan 15;61(1):57-70. doi: 10.1016/j.neuron.2008.11.012

Julia A. Kaye, Natalie C. Rose, Brett Goldsworthy, Andrei Goga, Noelle D. L'Etoile

Prolonged stimulation leads to specific and stable changes in an animal's behavior. In interneurons, this plasticity requires spatial and temporal control of neuronal protein synthesis. Whether such translational control occurs in sensory neurons is not known. Adaptation of the AWC olfactory sensory neurons of C. elegans requires the cGMP-dependent protein kinase EGL-4. Here, we show that the RNA-binding PUF protein FBF-1 is required in the adult AWC for adaptation. In the odor-adapted animal, it increases translation via binding to the egl-4 3' UTR. Further, the PUF protein may localize translation near the sensory cilia and cell body. Although the RNA-binding PUF proteins have been shown to promote plasticity in development by temporally and spatially repressing translation, this work reveals that in the adult nervous system, they can work in a different way to promote experience-dependent plasticity by activating translation in response to environmental stimulation.

In Caenorhabditis elegans, the AWC neurons are thought to deploy a cGMP signaling cascade in the detection of and response to AWC sensed odors. Prolonged exposure to an AWC sensed odor in the absence of food leads to reversible decreases in the animal's attraction to that odor. This adaptation exhibits two stages referred to as short-term and long-term adaptation. Previously, the protein kinase G (PKG), EGL-4/PKG-1, was shown necessary for both stages of adaptation and phosphorylation of its target, the beta-type cyclic nucleotide gated (CNG) channel subunit, TAX-2, was implicated in the short term stage. Here we uncover a novel role for the CNG channel subunit, CNG-3, in short term adaptation. We demonstrate that CNG-3 is required in the AWC for adaptation to short (thirty minute) exposures of odor, and contains a candidate PKG phosphorylation site required to tune odor sensitivity. We also provide in vivo data suggesting that CNG-3 forms a complex with both TAX-2 and TAX-4 CNG channel subunits in AWC. Finally, we examine the physiology of different CNG channel subunit combinations.

Prolonged odor exposure causes a specific, reversible adaptation of olfactory responses. A genetic screen for negative regulators of olfaction uncovered mutations in the cGMP-dependent protein kinase EGL-4 that disrupt olfactory adaptation in C. elegans. G protein-coupled olfactory receptors within the AWC olfactory neuron signal through cGMP and a cGMP-gated channel. The cGMP-dependent kinase functions in AWC neurons during odor exposure to direct adaptation to AWC-sensed odors, suggesting that adaptation is a cell intrinsic process initiated by cGMP. A predicted phosphorylation site on the beta subunit of the cGMP-gated channel is required for adaptation after short odor exposure, suggesting that phosphorylation of signaling molecules generates adaptation at early time points. A predicted nuclear localization signal within EGL-4 is required for adaptation after longer odor exposure, suggesting that nuclear translocation of EGL-4 triggers late forms of adaptation.

Neuron. 2000 Mar;25(3):575-86.

Noelle. D. L'Etoile, Cornelia I. Bargmann

Animals in complex environments must discriminate between salient and uninformative sensory cues. Caenorhabditis elegans uses one pair of olfactory neurons called AWC to sense many different odorants, yet the animal can distinguish each odorant from the others in discrimination assays. We demonstrate that the transmembrane guanylyl cyclase ODR-1 is essential for responses to all AWC-sensed odorants. ODR-1 appears to be a shared signaling component downstream of odorant receptors. Overexpression of ODR-1 protein indicates that ODR-1 can influence odor discrimination and adaptation as well as olfaction. Adaptation to one odorant, butanone, is disrupted by ODR-1 overexpression. Olfactory discrimination is also disrupted by ODR-1 overexpression, probably by overproduction of the shared second messenger cGMP. We propose that AWC odorant signaling pathways are insulated to permit odor discrimination.

PLoS Genet. 2016 Jul 26;12(7):e1006153. doi: 10.1371/journal.pgen.1006153. eCollection 2016 Jul.

Michelle C. Krzyzanowski, Sarah Woldemariam, Jordan F. Wood, Aditi H. Chaubey, Chantal Brueggemann, Alexander Bowitch, Mary Bethke, Noelle D. L’Etoile, Denise M. Ferkey 

All animals rely on their ability to sense and respond to their environment to survive. However, the suitability of a behavioral response is context-dependent, and must reflect both an animal's life history and its present internal state. Based on the integration of these variables, an animal's needs can be prioritized to optimize survival strategies. Nociceptive sensory systems detect harmful stimuli and allow for the initiation of protective behavioral responses. The polymodal ASH sensory neurons are the primary nociceptors in C. elegans. We show here that the guanylyl cyclase ODR-1 functions non-cell-autonomously to downregulate ASH-mediated aversive behaviors and that ectopic cGMP generation in ASH is sufficient to dampen ASH sensitivity. We define a gap junction neural network that regulates nociception and propose that decentralized regulation of ASH signaling can allow for rapid correlation between an animal's internal state and its behavioral output, lending modulatory flexibility to this hard-wired nociceptive neural circuit.

PLoS Genet. 2013;9(7):e1003619. doi: 10.1371/journal.pgen.1003619

Michelle C. Krzyzanowski, Chantal Brueggemann , Meredith J. Ezak, Jordan F. Wood, Kerry L. Michaels, Christopher A. Jackson, Bi-Tzen Juang, Kimberly D. Collins, Michael C. Yu, Noelle D. L'Etoile, Denise M. Ferkey 

Signaling levels within sensory neurons must be tightly regulated to allow cells to integrate information from multiple signaling inputs and to respond to new stimuli. Herein we report a new role for the cGMP-dependent protein kinase EGL-4 in the negative regulation of G protein-coupled nociceptive chemosensory signaling. C. elegans lacking EGL-4 function are hypersensitive in their behavioral response to low concentrations of the bitter tastant quinine and exhibit an elevated calcium flux in the ASH sensory neurons in response to quinine. We provide the first direct evidence for cGMP/PKG function in ASH and propose that ODR-1, GCY-27, GCY-33 and GCY-34 act in a non-cell-autonomous manner to provide cGMP for EGL-4 function in ASH. Our data suggest that activated EGL-4 dampens quinine sensitivity via phosphorylation and activation of the regulator of G protein signaling (RGS) proteins RGS-2 and RGS-3, which in turn downregulate Gα signaling and behavioral sensitivity

Predators and prey co-evolve, each maximizing their own fitness, but the effects of predator-prey interactions on cellular and molecular machinery are poorly understood. Here, we study this process using the predator Caenorhabditis elegans and the bacterial prey Streptomyces, which have evolved a powerful defense: the production of nematicides. We demonstrate that upon exposure to Streptomyces at their head or tail, nematodes display an escape response that is mediated by bacterially produced cues. Avoidance requires a predicted G-protein-coupled receptor, SRB-6, which is expressed in five types of amphid and phasmid chemosensory neurons. We establish that species of Streptomyces secrete dodecanoic acid, which is sensed by SRB-6. This behavioral adaptation represents an important strategy for the nematode, which utilizes specialized sensory organs and a chemoreceptor that is tuned to recognize the bacteria. These findings provide a window into the molecules and organs used in the coevolutionary arms race between predator and potential prey.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a severe neurodegenerative disorder that affects carriers of premutation CGG-repeat expansion alleles of the fragile X mental retardation 1 (FMR1) gene; current evidence supports a causal role of the expanded CGG repeat within the FMR1 mRNA in the pathogenesis of FXTAS. Though the mRNA has been observed to induce cellular toxicity in FXTAS, the mechanisms are unclear. One common neurophysiological characteristic of FXTAS patients is their inability to properly attenuate their response to an auditory stimulus upon receipt of a small pre-stimulus. Therefore, to gain genetic and cell biological insight into FXTAS, we examined the effect of expanded CGG repeats on the plasticity of the olfactory response of the genetically tractable nematode, Caenorhabditis elegans (C. elegans). While C. elegans is innately attracted to odors, this response can be downregulated if the odor is paired with starvation. We found that expressing expanded CGG repeats in olfactory neurons interfered with this plasticity without affecting either the innate odor-seeking response or the olfactory neuronal morphology. Interrogation of three RNA regulatory pathways indicated that the expanded CGG repeats act via the C. elegans microRNA (miRNA)-specific Argonaute ALG-2 to diminish olfactory plasticity. This observation suggests that the miRNA-Argonaute pathway may play a pathogenic role in subverting neuronal function in FXTAS.

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