Roux*, Antoine et al. (2021) International Worm Meeting "A single-cell expression atlas of C. elegans adulthood uncovers new aging trajectories"

Kelley, David, Hendrickson, David, Kerr, Rez, Podshivalova, Katie, Yuan*, Han, Kenyon, Cynthia, Roux*, Antoine, Paw, Jonathan

[International Worm Meeting, 2021]

Key aspects of aging biology remain mysterious. Does every tissue age at the same rate and in the same way? In this work, we applied the most recent advances in single-cell RNA sequencing in combination with a dissociation protocol to isolate worm cells in suspension (Zhang, Banerjee, and Kuhn, Plos One, 2011). Our goal was to address this question analyzing changes in gene expression regulation in every tissue across adulthood. Our final dataset contains 59,376 cells quantifying 24,297 genes at 6 ages ranging from day 1 of adulthood to day 15. We identified 211 unique expression states and matched these states to the majority of known cell types using systematic annotation. The quality of our cell identity annotation was validated by comparison to existing scRNAseq data and by using transgenic reporters in vivo. In young worms, we could assign functions to a few cell types whose expression was never accessible before. For instance, we observed that uv1 and uv3 vulva cells as well as GLR cell transcriptomes fell into neuronal categories. Moreover, we established a complete list of marker genes and candidate promoters for every cell type. We then analyzed the activities of 212 transcription factors (TFs) in every cell type, confirming some previously known TF activities (including elt-7, ces-1) and uncovering new ones (such as hlh-15, lin-32, klf-2, vab-15). The comparison across aging revealed a gene expression program that changes uniformly in the vast majority of cell types. This program includes genes previously found by bulk expression profiling (mitochondrial respiration, small heat shock proteins, vitellogenin and collagen). It also includes new transcription factors that appear to be functionally relevant. Interestingly, some critical GO-term gene-sets, like translation, nucleolus and proteasome have opposite aging trajectories among different tissues, suggesting that these tissues may age in fundamentally different ways. Our analysis also suggested that some tissues age faster than others, substantiated in vivo in preliminary experiments. All our data will be made publicly accessible online in a portal released upon publication.

Massimo A Hilliard et al. (2002) West Coast Worm Meeting "Intracellular Ca ++ increase in ASH sensory neuron in response to water-soluble chemical repellents."

William R Schafer, Alfonso J Apicella, Rex Kerr, Massimo A Hilliard, Paolo Bazzicalupo, HIROSHI SUZUKI

[West Coast Worm Meeting, 2002]

C.elegans is able to detect and avoid water-soluble chemical repellents in the environment. This behaviour is mainly mediated by few sensory neurons of the amphid, of which ASH is the principal player. We are interested in understanding the molecular mechanisms underlying the detection and transduction of the different chemical stimuli by ASH, as well as characterizing the activity of the neuronal circuit downstream the sensory cells. Recently, optical recordings of neuronal and muscle cell activity have been made in C.elegans using cameleon, a novel calcium sensor (Kerr et al. 2000). Cameleons are genetically-encoded fluorescent indicators for Ca++ in which a short-wavelength mutant of green fluorescent protein (GFP), calmodulin (CaM), a CaM-binding peptide (M13), and a long-wavelength mutant of GFP are tandemly fused. Binding of Ca++ to the CaM causes it to grab the M13 peptide, thus increasing FRET (fluorescence resonance energy transfer) from the short wavelength mutant GFP (donor) to the long-wavelength one (acceptor).

Massimo A Hilliard et al. (2002) European Worm Meeting "Sensory neuron Ca++ imaging in response to water-soluble chemical repellents."

Paolo Bazzicalupo, Christian Frokjar-Jensen, Rex Kerr, William R Schafer, Massimo A Hilliard, Alfonso J Apicella

[European Worm Meeting, 2002]

C.elegans is able to detect and avoid water-soluble chemical repellents in the environment and this behaviour is mainly mediated by few sensory neurons of the amphid of which ASH is the principal player. We are interested in understanding the molecular mechanisms underlying the detection and transduction of the different chemical stimuli as well as looking at the activity of the neuronal circuit downstream the sensory cells. Recently, optical recordings of neuronal and muscle cell activity have been made in C. elegans using a novel calcium sensor called cameleon (Kerr et al. 2000). Camaleons are genetically-encoded fluorescent indicators for Ca++ in which a shortwavelength mutant of green fluorescent protein (GFP), calmodulin (CaM), a CaM-binding peptide (M13), and a long-wavelength mutant of GFP are tandemly fused. Binding of Ca++ to the CaM causes it to grab the M13 peptide, thus increasing FRET (fluorescence resonance energy transfer) from the short wavelength mutant GFP (donor) to the long-wavelength one (acceptor). We decided to use this approach and we started by investigating the activity of the sensory cells. Using a cell specific promoter, we have generated lines expressing camaleon in the ASH sensory neuron (osm-10::YC2.3) and the Ca++ influx of this neuron has been imaged after stimulation with different chemical repellents including high osmotic strength, copper ions, SDS and quinine. As complementary strategy, we are producing cell cultures of the same transgenic strain to isolate in vitro ASH sensory neuron expressing camaleon and compare its responses to the selected repellents. The outcome of these experiments will be presented.

Rex Kerr et al. (2002) East Coast Worm Meeting "Cameleon-reported Insights into Touch Receptor Channel Function in Vivo"

Monica Driscoll, Dan Slone, William Schafer, Rex Kerr, HIROSHI SUZUKI, Jian Xue

[East Coast Worm Meeting, 2002]

Mechanical signaling, such as underlies the sense of touch, is the least understood type of signal transduction. Elegant genetic analyses of gentle body touch sensation conducted by Chalfie and colleagues have provided data supporting a molecular model of touch transduction. In this model, a candidate mechanically-gated touch-transducing channel (made up of MEC-4 and MEC-10 subunits of the DEG/ENaC channel superfamily) associates with additional MEC proteins inside and outside the cell. These contacts tether the channel to extracellular and intracellular proteins in associations that exert gating tension. A real challenge in testing the working model of touch transduction is in finding an appropriate system for electrophysiological testing of mechanosensitive gating. Expression in heterologous systems such as oocytes is problematic because several proteins, both inside and outside the cell, must contact the channel to deliver gating precise forces. Direct patching of touch neurons is also technically difficult given that tiny neurons are tightly tied to the cuticle within a specialized extracellular matrix. For this reason, we have been interested in in vivo assays of channel function, such as made possible by the work with gene-based sensors of neuronal activity such as calcium sensing cameleons. In previous work, Kerr et al. (Neuron 26:583-594) reported effective use of a cameleon to report transient calcium changes in muscle and neurons. The cameleon houses a YFP and a CFP connected by a calcium binding domain. When calcium is bound to the cameleon, a conformational change allows for fluorescence resonance energy transfer so that ratiometric measurements of YFP and CFP signals can be used to indicate changes in calcium concentrations.

DEVKOTA, RANJAN et al. (2015) International Worm Meeting "Novel regulators of the C. elegans UNC-6/Netrin pathway."

DEVKOTA, RANJAN, Garriga, Gian, Morck, Catarina, CHIEN, JASON

[International Worm Meeting, 2015]

Precise targeting of axons is essential for the proper functioning of the nervous system. Axonal growth cones respond to various guidance cues and navigate through a complex environment to reach their synaptic targets1. Even though many guidance cues and receptors have been identified, there is less knowledge of how axons respond to different cues in different phases of their growth. The aim of this project is to enhance the understanding of axon guidance by focusing on the development of the bilaterally symmetric Hermaphrodite Specific Neurons (HSNs). Each HSN extends a single axon to the ventral nerve cord, which then turns anteriorly and extends to the nerve ring2. The HSNs innervate vulval muscles and the AVF neurons to stimulate hermaphrodite egg laying and locomotion, respectively3,4. We have isolated three mutants whose HSN axons fail to extend ventrally. Our preliminary mapping, complementation and genetic interaction data suggest that these mutations define three genes that function together to antagonize the conserved UNC-6/Netrin signaling pathway. Our initial goal is to identify the underlying mutations with whole genome sequencing and rescue experiments. The identification of these genes will shed light on how the UNC-6/Netrin pathway is modulated for proper axon guidance along the dorsal-ventral axis.References:1. Dickson, B. J. Molecular mechanisms of axon guidance. Science 298, 1959-1964(2002).2. Garriga, G., Desai, C. & Horvitz, H. R. Cell interactions control the direction of outgrowth, branching and fasciculation of the HSN axons of Caenorhabditis elegans. Development 117, 1071-1087(1993).3. Desai, C., Garriga, G., McIntire, S. L. & Horvitz, H. R. A genetic pathway for the development of the Caenorhabditis elegans HSN motor neurons. Nature 336, 638-646(1988).4. Hardaker, L. A., Singer, E., Kerr, R., Zhou, G. & Schafer, W. R. Serotonin modulates locomotory behavior and coordinates egg-laying and movement in Caenorhabditis elegans. J. Neurobiol. 49, 303-313(2001). .

Alfonso Jr Apicella et al. (2003) International Worm Meeting "G-proteins and calcium release into the polymodal sensory neurons ASH."

Alfonso Jr Apicella, Rex Kerr, Massimo Hilliard, HIROSHI SUZUKI, Paolo Bazzicalupo, William R Scafer

[International Worm Meeting, 2003]

The C. elegans ASH sensory neurons are required to detect many water soluble repellants that taste bitter to humans, as well as high osmolarity and touch to the nose. It is unclear how sensory transduction occurs in these polymodal sensory neurons to allow detection and disdiscrimination of a diverse variety of aversive stimuli. To address this question we have we used the optical calcium indicator cameleon to measure neuronal responses to sensory stimuli in intact behaving animals (Kerr et al. Neuron2000). By locally perfusing repellants onto immobilized animals expressing cameleon in the ASH neurons, it has been possible to detect and measure calcium transients that accompany the excitation of these cells by sensory stimuli. The ASH neurons express multiple G-proteins, including at least 7 different subunits. We assayed the effect of three subunit genes, odr-3, gpa-1, and gpa-3, as well as one subunit gene, gpc-1, on repellant-activated calcium transients in ASH. Our results suggest that ODR-3 is involved in all ASH sensory responses, since the magnitudes of responses to all repellents tested were reduced in odr-3 mutants. In contrast, gpa-3 mutations affected only the detection of quinine, while gpa-1 mutations affected only glycerol responses. Thus, these G-proteins appear to participate in signaling pathways specific for particular repellants. Interestingly, gpa-1 and gpa-3 mutants (but not odr-3) were also abnormal in glycerol and quinine adaptation, respectively, suggesting that the repellant-specific signaling pathways are responsible for repellant adaptation and/or discrimination. gpc-1 mutants ( Jansen et all, EMBO Journal 2002) showed an adaptation-defective phenotype with respect to all repellants, suggesting that the G-protein multimers involved in adaptation all contain the GPC-1 subunit. Additional experiments addressing the roles of these molecules in sensory reponse and adaptation will be presented.

Mori I et al. (2000) Japanese Worm Meeting "An approach toward imaging activities of the neuronal network: a progress report 2000"

Mori I, Kimura KD

[Japanese Worm Meeting, 2000]

One of the big missing pieces for current neurobiology is to understand how a neuronal network works as a whole, instead of just a part of a big network. Apparently, our worm, C. elegans is the ideal system to solve the problem because, as well known, it has the description of its wiring diagram, many sophisticated molecular and genetic techniques, and so on. To monitor many neuronal activities in the network simultaneously, we have set up a system for calcium imaging of the worm neurons with cameleon, a GFP-based calcium sensor (ref. 1). Calcium changes in pharyngeal or body wall muscles have been monitored with cameleon (ref. 2 - 4). Our system is composed of W-View optics and HiSCA fast scan cooled CCD camera (Hamamatsu). The optics separates an image into two images based on spectrum information with dichroic mirror, and projects the two images to an imaging detector side by side. Thus, it enables simultaneous recording of two different fluorescence images from CFP (FRET donor) and YFP (acceptor) of cameleon. We expressed cameleon (YC2.1, ref. 5) specifically in AFD thermo-sensing neuron with gcy-8 promoter (a gift from B. Wedel and D. Garbers). The fluorescence intensities of CFP and YFP were reasonably strong, and we are trying to detect ratio change (YFP/CFP) during temperature change. We will report our strategy, results, and current problems. We hope this will help those who also want to apply the system to other aspects of the worm biology. References: 1. Miyawaki et al., Nature 388 (1997). 2. Kerr et al., 1999 IWM. 3. Centonzel et al., 1999 IWM. 4. Yu and Bargmann, 1999 IWM. 5. Miyawaki et al., PNAS 96 (1999).

Alfonso junior Apicella et al. (2004) West Coast Worm Meeting "C.elegans recognizes protons as a nociceptive stimulus through the DEG/EnaC and TRP channel."

Robert D Slone, William R Schafer, Alfonso junior Apicella, Monica Discroll

[West Coast Worm Meeting, 2004]

Acidic pH is known to activate both nociceptive neurons and acid taste chemosensory neurons in mammals. Previous experiments have shown that Caenorhabditis elegans also avoids an acidic environment (Sambongi et al. 2000). This avoidance behavior is dependent on multiple amphid chemosensory neurons, including the polymodal nociceptor ASH. The ASH sensory neurons are required to detect many water-soluble repellants that taste bitter to humans, as well as high osmolarity and touch to the nose. We have been interested in determining how pH sensation occurs in ASH, and how this sensory transduction process relates those involved in detecting other aversive stimuli. To address this question we have we used in vivo optical imaging with the calcium indicator cameleon (Kerr et al. Neuron 2000), together with behavioral experiments, to evaluate ASH sensory responses to acid pH and other stimuli. We observe robust calcium transients in ASH in response to stimulation with either acetic or hydrochloric acid; interestingly, after prolonged stimulation, we also observe a smaller "off" response following the return to neutral pH. In C.elegans members of the DEG/ENaC ion channel subunit superfamily (degenerin/epithelial Na+ channel) encode amiloride-sensitive sodium ion channels are implicated in mechanotransduction. The ASH neurons express three degenerin homologues: deg-1, C24G7.2, and ZK770.1 (Tavernarakis et al., IWM 662). By analyzing the effects of loss-of-function alleles of these genes, we determined that deg-1 and C24G7.2 function semi-redundantly in the detection of acidic pH; both single mutants exhibit abnormal acid-induced calcium influx in ASH, while the deg-1; C24G7.2 double mutant shows virtually no response to acid stimulation. Interestingly, deg-1 and C24G7.2 do not appear to affect ASH responses to chemical repellants such as copper; this contrasts with the ASH TRP channel OSM-9, which is required for all ASH sensory responses. Possible functional interactions between degenerin and TRP channels will be discussed.

Alfonso J Apicella et al. (2004) East Coast Worm Meeting "C.elegans recognizes protons as a nociceptive stimulus through the DEG/EnaC and TRP channel."

Monica Driscoll, Alfonso J Apicella, Robert D Slone, William R Schafer

[East Coast Worm Meeting, 2004]

Acidic pH is known to activate both nociceptive neurons and acid taste chemosensory neurons in mammals. Previous experiments have shown that Caenorhabditis elegans also avoids an acidic environment (Sambongi et al. 2000). This avoidance behavior is dependent on multiple amphid chemosensory neurons, including the polymodal nociceptor ASH. The ASH sensory neurons are required to detect many water-soluble repellants that taste bitter to humans, as well as high osmolarity and touch to the nose. We have been interested in determining how pH sensation occurs in ASH, and how this sensory transduction process relates those involved in detecting other aversive stimuli. To address this question we have we used in vivo optical imaging with the calcium indicator cameleon (Kerr et al. Neuron 2000), together with behavioral experiments, to evaluate ASH sensory responses to acid pH and other stimuli. We observe robust calcium transients in ASH in response to stimulation with either acetic or hydrochloric acid; interestingly, after prolonged stimulation, we also observe a smaller "off" response following the return to neutral pH. In C.elegans members of the DEG/ENaC ion channel subunit superfamily (degenerin/epithelial Na+ channel) encode amiloride-sensitive sodium ion channels are implicated in mechanotransduction. The ASH neurons express three degenerin homologues: deg-1 , C24G7.2, and ZK770.1 (Tavernarakis et al., IWM 662). By analyzing the effects of loss-of-function alleles of these genes, we determined that deg-1 and C24G7.2 function semi-redundantly in the detection of acidic pH; both single mutants exhibit abnormal acid-induced calcium influx in ASH, while the deg-1; C24G7.2 double mutant shows virtually no response to acid stimulation. Interestingly, deg-1 and C24G7.2 do not appear to affect ASH responses to chemical repellants such as copper; this contrasts with the ASH TRP channel OSM-9, which is required for all ASH sensory responses. Possible functional interactions between degenerin and TRP channels will be discussed.

Andrew Giles et al. (2008) C.elegans Neuronal Development Meeting "Validating a Multi-Worm Tracker for use in a Genetic Screen for Abnormal Habituation in C. elegans"

Nicholas Swierczek, Catharine Rankin, Andrew Giles, Rex Kerr

[C.elegans Neuronal Development Meeting, 2008]

Habituation is the gradual decrease in the behavioral response to an irrelevant stimulus. It is a fundamental form of learning in which organisms stop responding to stimuli that do not have significant consequences; this allows them to reserve their energy to respond to stimuli that predict biologically relevant consequences. The cellular and molecular mechanisms that mediate this form of learning are not well-understood. C. elegans can be used as a model to study this because their response to a non-localized tap (called the tap withdrawal response) has been shown to habituate (Rankin et al. 1990). Conducting a large scale genetic screen would be beneficial in order to identify a number of novel genes that play a role in habituation. Until now this has been a daunting task because it takes a long time to analyze an individual strain (approx. 40 hours by manual behavioural analysis; 6 hours using an automated system that tracks individual worms). The Kerr lab has developed a system that is capable of simultaneously analyzing the habituation of multiple worms (as many as 50 reliably) in real-time. This lowers the time needed to assess an individual strain to 10 minutes, making a genetic screen for habituation feasible. Prior to conducting a screen it is important to validate the system to be sure it will properly identify habituation abnormalities. We have validated the system by demonstrating a number of essential habituation characteristics, such as the interstimulus interval dependence on both the rate and asymptotic level of habituation in wild-type worms (Rankin and Broster 1992). We have also tested several previously identified mutants, such as dop-1 and cat-2, which show altered short-term habituation (Sanyal et al. 2004) and found that the system is capable of reproducing the same phenotypes as previously reported. We are currently optimizing the system for use in a genetic screen to identify genes that affect different characteristics of habituation.