Massimo A Hilliard et al. (2002) Worm Breeder's Gazette "qui-1 has been molecularly identified and corresponds to Y45F10B.10"

Massimo A Hilliard, Ronald HA Plasterk, Paolo Bazzicalupo

[Worm Breeder's Gazette, 2002]

We have previously described the isolation, after EMS mutagenesis, of a series of mutants that have lost the ability to avoid the water soluble repellent quinine hydrocloride ( qui mutants=QUInine non-avoiders). Five mutants presented an unaltered response to light touch and a wild-type structure of the sensory cilia when stained with the fluorescent dye DiO. One of them, qui-1 ( gb 404 ) showed very low avoidance of quinine and SDS although its avoidance responses to high osmotic strength and Cu ++ appear unchanged. We cloned qui-1 using the SNPs mapping strategy (Wicks et al. 2001). Initially, we positioned qui-1 in a small interval on the LG IV of about 120 kb, between the two SNPs Y45F10A and C08F11A. Then, we rescued the quinine avoidance mutant phenotype by injecting long wild-type PCR products in the qui-1 ( gb404 ) strain. The smallest rescuing fragment was 11 kb and contained a single gene Y45F10B.10, predicted by the C.elegans genome-sequencing consortium. We then sequenced the complete Y45F10B.10 gene in the qui-1 ( gb404) strain and found a CAA to TAA transition that generates a stop codon in the first exon. The locus can in principle produce a truncated protein of only 76 aa which presumably has no function. The results obtained in the rescue experiments and those of the sequence of the qui-1 ( gb404 ) indicate that qui-1 corresponds to Y45F10B.10. BLAST search results revealed that QUI-1 is a novel protein of unknown function that contains four WD 40 domains described as functional modules for protein-protein interaction. However the cellular function of QUI-1 cannot, at present, be inferred from its amino acid sequence. Using different GFP fusion constructs we discovered that qui-1 is expressed in a subset of the sensory neurons of the amphid including ASH, ADL that have been previously identified as water soluble avoidance neurons. Using GFP constructs with the entire gene we found that the localization of the protein is not confined to the sensory cilia instead the GFP is diffuse in the cell body, the dendrite, sensory cilia and axon. References: Wicks SR, Yeh RT, Gish WR, Waterston RH, Plasterk RHA. (2001). Rapid gene mapping in Caenorhabditis elegans using a high density polymorphism map. Nature Genetics 28: 160-164.

Valperga G et al. (2022) Elife "Impairing one sensory modality enhances another by reconfiguring peptidergic signalling in Caenorhabditis elegans."

De Bono M, Valperga G

[Elife, 2022]

Animals that lose one sensory modality often show augmented responses to other sensory inputs. The mechanisms underpinning this cross-modal plasticity are poorly understood. We probe such mechanisms by performing a forward genetic screen for mutants with enhanced O₂ perception in <i>Caenorhabditis elegans</i>. Multiple mutants exhibiting increased O₂ responsiveness concomitantly show defects in other sensory responses. One mutant, <i>qui-1</i>, defective in a conserved NACHT/WD40 protein, abolishes pheromone-evoked Ca²⁺ responses in the ADL pheromone-sensing neurons. At the same time, ADL responsiveness to pre-synaptic input from O₂-sensing neurons is heightened in <i>qui-1</i>, and other sensory defective mutants, resulting in enhanced neurosecretion although not increased Ca²⁺ responses. Expressing <i>qui-1</i> selectively in ADL rescues both the <i>qui-1</i> ADL neurosecretory phenotype and enhanced escape from 21% O₂. Profiling ADL neurons in <i>qui-1</i> mutants highlights extensive changes in gene expression, notably of many neuropeptide receptors. We show that elevated ADL expression of the conserved neuropeptide receptor NPR-22 is necessary for enhanced ADL neurosecretion in <i>qui-1</i> mutants, and is sufficient to confer increased ADL neurosecretion in control animals. Sensory loss can thus confer cross-modal plasticity by changing the peptidergic connectome.

Bethany L Johanson et al. (2004) West Coast Worm Meeting "Mapping two Suppressors of the pharyngeal morphogenesis mutant, pha-1"

David S Fay, Bethany L Johanson, Xiaohui Qui

[West Coast Worm Meeting, 2004]

lin-35 , a gene originally identified as a class B SynMuv gene, is the sole member of the Retinoblastoma protein (Rb) family in C. elegans. Numerous studies in multiple systems, including C. elegans (Boxem and van den Heuvel, 2001; Fay et al., 2002), have shown Rb to be an important regulator of cell cycle progression. To discover additional functions for Rb family proteins in C. elegans, we have carried out screens to identify mutations that show synthetic interactions with lin-35 loss of function (LOF; Fay et al 2002). Among the isolated alleles were partial LOF mutations ubc-18 , a ubiquitin conjugating enzyme, and pha-1 , a gene of unknown function (Granato et al., 1994; Fay et al., 2003, 2004). Both mutations lead to early defects in pharyngeal morphogenesis in conjunction with lin-35 LOF . We also observed a strong genetic interaction between lin-35 and a previously isolated temperature sensitive allele of pha-1, e2123 (Schnabel et al., 1990). In addition, we found that ubc-18 and pha-1 were themselves synthetically lethal, and that two previously isolated suppressors of pha-1 , sup-36 and sup-37 , could suppress the synthetic lethality of lin-35; pha-1 and lin-35; ubc-18 mutants. The mutual suppression of lin-35; ubc-18 and pha-1 by the suppressors provides further evidence that LIN-35, UBC-18, and PHA-1 function in parallel pathways and may act on a common target. To gain insight into the cause of the synthetic lethality, and to uncover novel targets for these proteins, we are cloning both suppressor mutations. sup-36 has been mapped using genetic and SNP methods to a ~1Mb region on linkage group (LG) IV while sup-37 has been narrowed down to a ~800 kb region on linkage group (LG) V. We will present our mapping data and progress on the analysis of these mutants. Boxem and van den Heuvel. Development. 128(21): 4349-59, 2001 Fay et al. Genes & Development. 16: 503-517, 2002 Fay et al. Development. 130(14): 3319-30, 2003 Fay et al. Developmental Biology. 271: 11-25, 2004 Granato et al. Development. 120(10): 3005-17, 1994 Schnabel H and Schnabel R. Science. 250: 686, 1990 Schnabel et al. Genetics. 129: 69-77, 1991

Felix MA et al. (2002) Hermann, Editeurs des Sciences et des Arts. Paris, France. "Caenorhabditis elegans. Un organisme modele en biologie."

Labouesse M, Segalat L, Felix MA

[Hermann, Editeurs des Sciences et des Arts. Paris, France., 2002]

L'espce Caenorhabditis elegans fut dcrite en 1900 Alger par E. Maupas, qui s'intressait son mode de reproduction hermaphrodite. Plus tard, vers le milieu du vingtime sicle, V. Nigon et ses collaboratuers Lyon tudirent les reorganizations cellulaires accompagnant la fecundation et les premiers clivages. J. Brun isola les preiers mutants morpholgiques.

Carmela Bergamasco et al. (2005) International Worm Meeting "Quinine avoidance: cells and molecules involved"

Carmela Bergamasco, Giovanni Esposito, Paolo Bazzicalupo

[International Worm Meeting, 2005]

We are interested in elucidating the cellular and molecular basis of the avoidance behavior generated by repellents in C. elegans. Among the repellents we particularly focused on quinine which is poisonous for cells and organisms and is perceived as bitter by humans. ASH is the main sensory neuron mediating quinine avoidance. To identify the molecules that, inside ASH, are necessary for sensing quinine we are following two approaches, the identification of new genes with forward screens and a best candidate approach. In the first one, to obtain new quinine-non-avoider mutants we conducted a clonal screen after EMS mutagenesis. We screened single F2 worms using the drop test (Hilliard et al., 2002). This screen allows one to identify non-avoider mutants largely independently of other influences (adaptation, defective movement, social behavior, etc.). We identified 17 new mutants. Two of them do not complement grk-2 (rt97) which has been shown to mediate also octanol avoidance (Fukuto et al., 2004) and three of the mutants, do not complement qui-1 (gb404), a gene identified in a previous screen and directly involved in quinine avoidance (Hilliard et al., 2004). QUI-1 is a novel protein containing in N-terminal region a Nacht Domain, (a predicted nucleoside triphosphatase (NTPase) domain) and in C-terminal region 12 WD repeats. Expression studies based on reporters indicate that it is expressed in the ASH and ADL sensory neurons, which we know are involved in sensing quinine and other repellents. Using cell specific promoter, we showed that expression of QUI-1 in ASH is sufficient to rescue the quinine avoidance phenotype. To begin to understand the function of qui-1 we are carrying out genetic experiments to explore its role in quinine signalling; we have obtained specific antibodies and are using them to identify QUI-1 cellular localization; finally we are performing two hybrid experiments to identify some of its partners. In the second approach we have begun to test if mutants in genes involved in sensory signal transduction are involved in quinine signalling. We had already determined that two Ga proteins, GPA-3 and ODR-3, (Hilliard et al. 2004.) are involved in quinine avoidance. With different approaches we have also studied the role in the avoidance response of osm-9, egl-19 and gpc-1 (Hilliard et al., 2005). Thus, together with QUI-1 and GRK-2 we have identified several players acting within ASH to mediate avoidance and are trying to come up with a reasonable and testable model to explain their interaction and functioning.

C Bergamasco et al. (2004) European Worm Meeting "QUI-1 IS NECESSARY TO DEFINE THE CHEMOSENSORY FUNCTION OF THE NEURONS OF C. ELEGANS"

C Bergamasco, M Hilliard, P Bazzicalupo

[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.

Hilliard MA et al. (2004) EMBO J "Worms taste bitter: ASH neurons, QUI-1, GPA-3 and ODR-3 mediate quinine avoidance in Caenorhabditis elegans."

Hilliard MA, Plasterk RHA, Bazzicalupo P, Arbucci S, Bergamasco C

[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.

Carmela Bergamasco et al. (2006) European Worm Meeting "Signalling in quinine avoidance"

Paolo Bazzicalupo, Giovanni Esposito, Carmela Bergamasco, Salvatore Arbucci

[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.

Neal SJ et al. (2016) G3 (Bethesda) "A Forward Genetic Screen for Molecules Involved in Pheromone-Induced Dauer Formation in Caenorhabditis elegans."

Butcher RA, Sengupta P, DiTirro D, Park J, Neal SJ, Yoon J, Shibuya M, Kim K, Schroeder FC, Choi W

[G3 (Bethesda), 2016]

Animals must constantly assess their surroundings and integrate sensory cues to make appropriate behavioral and developmental decisions. Pheromones produced by conspecific individuals provide particularly critical information regarding environmental conditions. Ascaroside pheromone concentration and composition are instructive in the decision of C. elegans to either develop into a reproductive adult or enter into the stress-resistant alternate dauer developmental stage. Pheromones are sensed by a small set of sensory neurons, and integrated with additional environmental cues, to regulate neuroendocrine signaling and dauer formation. To identify molecules required for pheromone-induced dauer formation, we performed an unbiased forward genetic screen and identified phd (pheromone response defective dauer) mutants. Here we describe new roles in dauer formation for previously identified neuronal molecules such as the WD40 domain protein QUI-1 and MACO-1 Macoilin, report new roles for nociceptive neurons in modulating pheromone-induced dauer formation, and identify tau tubulin kinases as new genes involved in dauer formation. Thus, phd mutants define loci required for detection, transmission, or integration of pheromone signals in the regulation of dauer formation.

Hilliard MA et al. (1997) International C. elegans Meeting "C.ELEGANS MUTANTS THAT FAIL TO AVOID QUININE"

Bazzicalupo P, Hilliard MA

[International C. elegans Meeting, 1997]

We are studying chemoreception in C.elegans focusing our attention on the avoidance of water soluble substances. We have used a new test, "drop test", which consists in delivering a small drop of the chosen solution behind a worm freely moving on the plate surface. By capillarity the solution reaches the anterior end of the animal where the amphids are located. If the solution contains a repellent, the worm stops immediately and moves backward for a few seconds; if no repellent is present the worm continues its forward movement. Using this test we have identified, including those already known, 16 chemicals acting as repellent at concentrations between 0.1 and 10 mM. We have chosen quinine, at a concentration of 10 mM, to screen the F2 progeny of EMS mutagenized worms. Out of 70.000 worms screened we have found 15 mutants which fail to avoid quinine. After preliminary behavioral characterization we have focused our attention on two, gb404 and gb408. These are allelic and identify a new gene, qui-1, which has been positioned on the right arm of the LG IV using STS mapping. The amphid neurons in both alleles stain normally with FITC. They respond normally to light touch and to some other repellent e.g. copper ions, zinc ions, veratridin, levamisole, garlic extract, high osmotic strenght. We hope we have identified either the receptor for quinine, if it exists, or some other component acting relatively early in the sensory transduction pathway used by C.elegans to avoid quinine.