[
Cell Mol Life Sci,
2006]
The small nematode Caenorhabditis elegans lives in the soil, where mechanical, thermal and most of all chemical stimuli strongly influence its behavior. Here we briefly review how chemical sensitivity is organized at the cellular and molecular level in this organism. C. elegans has less than 40 chemosensory neurons. With few exceptions each neuron senses more than one substance and each substance is sensed by more than one neuron. At the molecular level, as in other organisms, also in C. elegans, seven transmembrane G-protein-coupled receptors (GPCRs), heterotrimeric G proteins, cyclic nucleotide- gated ion channels, TRP channels and Ca(++) play crucial roles in chemical sensitivity. An unusual feature, possibly due to C. elegans''s strong dependence on chemical cues, is the very large number of GPCR chemoreceptor genes (1300-1700) coded in its genome. Genetic approaches have also allowed the identification of new molecules involved in chemical sensitivity that would not have been discovered otherwise. In addition to the basic factors involved in primary signalling, the studies in C. elegans have revealed a network of regulatory pathways and molecules suggesting that fine modulation of the responsiveness of neurons is important, possibly to allow worms to negotiate a continuously changing environment. The experimental versatility of C. elegans has made it possible, in many cases, to determine precisely in which neuron a given molecule or pathway is required and for which biological response. This type of information can contribute to the general field of sensory signalling because it provides correlations between the biochemical properties of molecules and their cellular functions and between these and the in vivo behavioral responses of the animal.
[
European Worm Meeting,
2004]
The chemical enviroment has a crucial role in animal biology: nutrients attract organisms, pheromones induce sexual behavior, the detection of toxic or noxious substances leads to avoidance behavior. C.elegans exhibits an escape/avoidance reaction when it senses chemical stimuli which are toxic or dangerous. Quinine has various pharmacological effects and is generally poisonous for cells and organisms. During evolution humans have come to perceive it as bitter and C. elegans has learned to avoid it. We are interested in the mechanisms used by the worm to sense quinine in the environment. The polymodal sensory neuron ASH is the main sensory neuron for the detection of quinine. Other sensory neurons (ADL, ASK, and PHA and PHB) are also involved, but with a minor or modulating role (Hilliard et al., 2002; Current Biology). With regard to the molecules that, inside the sensory cells, are necessary for sensing quinine we are following two approaches. In a first one, we tested mutants in known chemoreception genes and found that the two G-alpha subunits, GPA-3 and ODR-3 are involved in quinine signaling. In a second approach, we screened directly for quinine-non-avoider mutants. Of 11 mutants showing no cilia defects and proper response to light touch, 4 identify the new gene
qui-1. QUI-1 is a novel protein
of1592 aa containing 12 repetitions of the WD40 domain. It is expressed in the sensory neurons ASH and ADL. Mammalian orthologs of
qui-1 and of a second C. elegans gene with a similar structure, have been identified (Hilliard et al. 2004 EMBO). To understand the function of
qui-1 we are studying its genetic interaction with known chemoreception genes. We are focusing on genes required for cilium structure, signal transduction, cation channels and neurotrasmitter release involved in sensory responses. The results of these genetic interactions will be presented.
[
Gene,
2007]
The nematode C. elegans has become an important model for understanding how genes influence behavior. However, in this organism the available approaches for identifying the neuron(s) where the function of a gene is required for a given behavioral trait are time consuming and restricted to non essential genes for which mutants are available. We describe a simple reverse genetics approach for reducing, in chosen C. elegans neurons, the function of genes. The method is based on the expression, under cell specific promoters, of sense and antisense RNA corresponding to a gene of interest. By targeting the genes
osm-10,
osm-6 and the Green Fluorescent Protein gene, gfp, we show that this approach leads to efficient, heritable and cell autonomous knock-downs of gene function, even in neurons usually refractory to classic RNA interference (RNAi). By targeting the essential and ubiquitously expressed gene,
gpb-1, which encodes a G protein beta subunit, we identify for the first time two distinct sets of neurons in which the function of
gpb-1 is required to regulate two distinct behaviors: egg-laying and avoidance of repellents. The cell specific knock-downs obtained with this approach provide information that is complementary to that provided by the cell specific rescue of loss-of-function mutations and represents a useful new tool for dissecting the role that genes play in selected neurons.
[
EMBO J,
2004]
An animal's ability to detect and avoid toxic compounds in the environment is crucial for survival. We show that the nematode Caenorhabditis elegans avoids many water-soluble substances that are toxic and that taste bitter to humans. We have used laser ablation and a genetic cell rescue strategy to identify sensory neurons involved in the avoidance of the bitter substance quinine, and found that ASH, a polymodal nociceptive neuron that senses many aversive stimuli, is the principal player in this response. Two G protein alpha subunits GPA-3 and ODR-3, expressed in ASH and in different, nonoverlapping sets of sensory neurons, are necessary for the response to quinine, although the effect of
odr-3 can only be appreciated in the absence of
gpa-3. We identified and cloned a new gene,
qui-1, necessary for quinine and SDS avoidance.
qui-1 codes for a novel protein with WD-40 domains and which is expressed in the avoidance sensory neurons ASH and ADL.
[
BMC Biol,
2010]
BACKGROUND: Polymodal, nociceptive sensory neurons are key cellular elements of the way animals sense aversive and painful stimuli. In Caenorhabditis elegans, the polymodal nociceptive ASH sensory neurons detect aversive stimuli and release glutamate to generate avoidance responses. They are thus useful models for the nociceptive neurons of mammals. While several molecules affecting signal generation and transduction in ASH have been identified, less is known about transmission of the signal from ASH to downstream neurons and about the molecules involved in its modulation. RESULTS: We discovered that the regulator of G protein signalling (RGS) protein, EGL-10, is required for appropriate avoidance responses to noxious stimuli sensed by ASH. As it does for other behaviours in which it is also involved,
egl-10 interacts genetically with the G(o)/(i) protein GOA-1, the G(q) protein EGL-30 and the RGS EAT-16. Genetic, behavioural and Ca(+) imaging analyses of ASH neurons in live animals demonstrate that, within ASH, EGL-10 and GOA-1 act downstream of stimulus-evoked signal transduction and of the main transduction channel OSM-9. EGL-30 instead appears to act upstream by regulating Ca(+) transients in response to aversive stimuli. Analysis of the delay in the avoidance response, of the frequency of spontaneous inversions and of the genetic interaction with the diacylglycerol kinase gene,
dgk-1, indicate that EGL-10 and GOA-1 do not affect signal transduction and neuronal depolarization in response to aversive stimuli but act in ASH to modulate downstream transmission of the signal. CONCLUSIONS: The ASH polymodal nociceptive sensory neurons can be modulated not only in their capacity to detect stimuli but also in the efficiency with which they respond to them. The G and RGS molecules studied in this work are conserved in evolution and, for each of them, mammalian orthologs can be identified. The discovery of their role in the modulation of signal transduction and signal transmission of nociceptors may help us to understand how pain is generated and how its control can go astray (such as chronic pain) and may suggest new pain control therapies.
[
European Worm Meeting,
2006]
Chemical sensitivity allows animals to identify and respond appropriately to the chemical composition of the environment. Noxious water-soluble compounds that are avoided by C. elegans are generally sensed as bitter by humans and are discarded in double choice test by mice. We have used C. elegans to focus on the molecular mechanisms involved in primary sensing of quinine, a molecule detected as bitter by humans. ASH is the main sensory neuron involved in sensing quinine. Two G? subunits, GPA-3 and ODR-3 are necessary for the response of ASH to repellent stimuli (Hilliard et al., 2004 and 2005). In addition the TRPV channel proteins, OSM-9 and OCR-2, are also necessary for the ASH avoidance responses (Colbert et al 1997, Tobin et al 2002). Finally we identified a novel protein, QUI-1, as an essential components of the response to quinine (Hilliard et al., 2004). With regard to the molecular function of QUI-1, we demonstrate that QUI-1 function is required in ASH for the response to quinine and, using specific antibodies, that the protein is localized to the sensory cilia. These results, together with the discovery that QUI-1 contains an RGS (Regulator of G protein Signaling) domain, strongly suggest that this novel protein might be involved in quinine signaling.. Are there other components of the quinine signal transduction pathway?. We are using a best candidate approach and a variety of behavioral assays to identify new molecules involved in sensing repellent chemicals and in particular quinine. We analyzed behaviorally loss of function and overexpression mutants in several molecules known to act in the G protein signaling pathways (G? subunits, G? subunits, RGS proteins, etc.). The results obtained will be discussed.. Colbert, H. A., Smith, T. L. and Bargmann, C. I. (1997). OSM-9, a novel protein with structural similarity to channels, is required for olfaction, mechanosensation, and olfactory adaptation in Caenorhabditis elegans. J Neurosci 17, 8259-69.. Hilliard, M. A., Apicella, A. J., Kerr, R., Suzuki, H., Bazzicalupo, P. and Schafer, W. R. (2005). In vivo imaging of C. elegans ASH neurons: cellular response and adaptation to chemical repellents. Embo J 24, 63-72.. Hilliard, M. A., Bergamasco, C., Arbucci, S., Plasterk, R. H. and Bazzicalupo, P. (2004). Worms taste bitter: ASH neurons, QUI-1, GPA-3 and ODR-3 mediate quinine avoidance in Caenorhabditis elegans. Embo J 23, 1101-11.. Tobin, D., Madsen, D., Kahn-Kirby, A., Peckol, E., Moulder, G., Barstead, R., Maricq, A. and Bargmann, C. (2002). Combinatorial expression of TRPV channel proteins defines their sensory functions and subcellular localization in C. elegans neurons. Neuron 35, 307-18.
[
BMC Genomics,
2007]
ABSTRACT: BACKGROUND: In the genome of Caenorhabditis elegans, homopolymeric poly-G/poly-C tracts (G/C tracts) exist at high frequency and are maintained by the activity of the DOG-1 protein. The frequency and distribution of G/C tracts in the genomes of C. elegans and the related nematode, C. briggsae were analyzed to investigate possible biological roles for G/C tracts. RESULTS: In C. elegans, G/C tracts are distributed along every chromosome in a non-random pattern. Most G/C tracts are within introns or are close to genes. Analysis of SAGE data showed that G/C tracts correlate with the levels of regional gene expression in C. elegans. G/C tracts are over-represented and dispersed across all chromosomes in another Caenorhabditis species, C. briggase. However, the positions and distribution of G/C tracts in C. briggsae differ from those in C. elegans. Furthermore, the C. briggsae
dog-1 ortholog CBG19723 can rescue the mutator phenotype of C. elegans
dog-1 mutants. CONCLUSIONS: The abundance and genomic distribution of G/C tracts in C. elegans, the effect of G/C tracts on regional transcription levels, and the lack of positional conservation of G/C tracts in C. briggsae suggest a role for G/C tracts in chromatin structure but not in the transcriptional regulation of specific genes.