Hiroko Ito et al. (2004) East Asia Worm Meeting "Genetic analysis of guanylyl cyclases and a cyclic nucleotide-gated cation channel involved in thermosensation"

John Satterlee, Piali Sengputa, Hitoshi Inada, Ikue Mori, Hiroko Ito

[East Asia Worm Meeting, 2004]

C. elegans senses temperature mainly by a pair of sensory neurons (AFD) and shows a characteristic behavior towards the temperature called thermotaxis. Several mutants that are defective in thermotaxis have been isolated, and these mutants can be divided into three classes: cryophilic (cold-seeking), thermophilic (heat-seeking), and athermotactic (non-temperature responsive) phenotype. Analyses of these mutants identified several genes involved in thermotaxis. tax-4 encoding a cyclic nucleotide-gated cation channel (CNG channel) is essential for thermosensation in AFD. tax-4 is also required for olfaction by sensory neuron AWC, implicating similar components in thermosensory and olfactory signal transduction. In AFD, three guanylyl cyclase genes, gcy-8, gcy-18 and gcy-23 , play an essential role for thermosensation (see abstract by Inada et al .). Analysis of eat-16 mutant, which is defective in a regulator of G protein signaling (RGS), revealed that AWC olfactory neuron functions as a thermosensory neuron and that odr-1 guanylyl cyclase is involved in thermosensory signal transduction in AWC (Okumura et al ., submitted). To investigate genetic interaction of these four guanylyl cyclases and TAX-4 CNG channel, we constructed tax-4;gcy-23 gcy-8 gcy-18 and gcy-23 gcy-8 gcy-18;odr-1 quadruple mutants, and examined their thermotaxis phenotype. While gcy-23 gcy-8 gcy-18 mutants showed cryophilic and athermotactic phenotype, tax-4;gcy-23 gcy-8 gcy-18 mutants showed athermotactic phenotype like tax-4 mutants, suggesting that tax-4 is epistatic to gcy-8, gcy-18 and gcy-23 . gcy-23 gcy-8 gcy-18;odr-1 mutants showed athermotactic phenotype, although odr-1 single mutants showed almost normal thermotactic phenotype. This result suggests that ODR-1, which is an essential component in olfactory signal transduction, functions in thermosensory signal transduction in AWC. Our results are consistent with a molecular model in which three guanylyl cyclases (GCY-8, GCY-18 and GCY-23) and ODR-1 function upstream of TAX-4 CNG channel for thermosensation in AFD and AWC, respectively.

Anthony A Ferrante et al. (2002) West Coast Worm Meeting "Check out my Profile! Isolation of chemotaxis defective mutants with altered str-1 expression levels using automated, high-sensitivity fluorescence profiling."

Gregory O'Connor, Steven Alam, Bruce Holcombe, Anthony A Ferrante, Jennifer Kean, Peter Van Osta, Vance Chang, Britta Moellers

[West Coast Worm Meeting, 2002]

Autofluorescence background, the natural fluorescence from biological materials, reduces sensitivity of detection of localized fluorescence in multicellular organisms. For example in the case of a transgenic C. elegans that expresses GFP in two cells, that fluorescence can be masked by the autofluorescence of the remaining 1000 somatic cells. We have solved this problem with an add-on to our COPAS analysis and sorter technology that allows the instrument to measure and store the pattern of fluorescence along the length of the animal. That information was used to restrict fluorescence analysis to the region of the animal that contained the fluorescent cells thus improving the signal to autofluorescence ratio more than 20-fold. We used the Profiler system to perform a genetic screen in which we isolated mutants of PY1089 (kindly provided by Piali Sengupta), a transgenic C. elegans that expresses GFP in the AWB sensory neurons under control of the str-1 promoter. Proper expression of the odorant receptor, STR-1, in the AWB sensory neurons is required for appropriate chemotactic response to the repulsive odorant 2-nonanone. Without the Profiler technology the COPAS system was unable to distinguish PY1089 from N2 wild type animals, and it could not be used in a genetic screen for mutants with reduced GFP expression. With the addition of Profiler we were able to clearly distinguish PY1089 from N2 and were further able to sort animals that had either reduced or increased GFP expression in the sensory neurons.

Chou JH et al. (1996) West Coast Worm Meeting "MOLECULAR ANALYSIS OF A PUTATIVE OLFACTORY RECEPTOR IN C. ELEGANS"

Bargmann CI, Chou JH, Sengupta P

[West Coast Worm Meeting, 1996]

The olfactory system is remarkable in its ability to couple broad sensitivity with high specificity. A long-standing question has been the nature of the molecular receptors that mediate this response. Although olfactory receptors have been identified in vertebrates, functional data linking receptors with physiological ligands has been lacking. Using C. elegans as a model system, we have identified a potential olfactory receptor with a candidate ligand. Using a behavioral screen, Piali Sengupta identified a mutant, odr-10(ky32), whose sole defect was an inability to respond to the volatile odorant diacetyl. Cloning odr-10 revealed that it belongs to the seven transmembrane domain superfamily of G-protein coupled receptors, with distant homology to rat olfactory receptors. odr-10 was expressed exclusively in AWA, the single chemosensory neuron required for diacetyl response. These findings implied that odr-10 might be the diacetyl receptor (Sengupta et al., Cell (1996) 84: 899-909). odr-10(ky32) is due to a missense mutation. Might the missense mutation affect only the diacetyl recognition site of a receptor that senses multiple compounds? Using the Tc1 insertion / imprecise excision strategy, a null mutant was identified in which a large part of the odr-10 gene was deleted. The null phenotype was also restricted to diacetyl, with responses to all other odorants normal. Thus, odr-10 is specifically required for diacetyl chemosensation. I am using this putative olfactory receptor and its candidate ligand to analyze olfactory function in vivo and possibly in vitro. Treatment with high concentrations of one odorant can abolish response to that odorant without affecting responses to other odorants -- how might this adaptation be mediated? What specifies the dose-response sensitivity of a receptor? What are the downstream effectors of the receptor? To address these questions, I am expressing both wild-type and modified forms of the odr-10 gene in C. elegans and in vertebrate cells.

Anthony A Ferrante et al. (2002) East Coast Worm Meeting "Check out my Profile! Isolation of mutants with altered str-1 expression levels using automated, high-sensitivity fluorescence profiling"

Britta Moellers, Bruce Holcombe, Steve Alam, Vance Chang, Anthony A Ferrante

[East Coast Worm Meeting, 2002]

Autofluorescence background, the natural fluorescence from biological materials, reduces sensitivity of detection of localized fluorescence in multicellular organisms. For example in the case of a transgenic C. elegans that expresses GFP in two cells, that fluorescence can be masked by the autofluorescence of the remaining 1000 somatic cells. We have solved this problem with an add-on to our COPAS analysis and sorter technology that allows the instrument to measure and store the pattern of fluorescence along the length of the animal. That information was used to restrict fluorescence analysis to the region of the animal that contained the fluorescent cells thus improving the signal to autofluorescence ratio more than 20-fold. We used the system to perform a genetic screen in which we isolated mutants of PY1089 (kindly provided by Piali Sengupta), a transgenic C. elegans that expresses GFP in the AWB sensory neurons under control of the str-1 promoter. Without the Profiler technology the COPAS system was unable to distinguish PY1089 from N2 wild type animals, and it could not be used in a genetic screen for mutants with reduced GFP expression. With the addition of Profiler we were able to clearly distinguish PY1089 from N2 and were further able to sort animals that had either reduced or increased GFP expression in the sensory neurons. F2 progeny of mutagenized PY1089 were analyzed and sorted using the COPAS BIOSORT with prototype Profiler hardware. First, 100 animals were run and their peak fluorescence intensities were determined. That information was used to set a range of normal peak heights in the Profiler. The Profiler was instructed to dispense those animals whose peak heights were outside of the normal range, either higher or lower, one per well into 96-well plates. Mutants were confirmed both by regrowing the sorted animals and running them through the Profiler a second time and visually by microscopy. Approximately 50,000 animals from 8 separate pools were screened and 1,000 were dispensed to wells in the first pass. We have retested 96 lines from the sorted animals to date. We have isolated 4 independent mutant lines with decreased GFP expression, 3 independent lines with increased GFP expression and 2 lines with a mix of increased, wild-type, and decreased GFP expression. We are in the process of further characterizing the mutants by fluorescence microscopy and we will further characterize the mutants for their chemotactic response to 2-nonanone, which is dependent on proper AWB neuronal function.

Anthony A Ferrante et al. (2002) Mid-west Worm Meeting "Check out my Profile! Isolation of chemotaxis defective mutants with altered str-1 expression levels using automated, high-sensitivity fluorescence profiling"

Bruce Holcombe, Peter Van Osta, Vance Chang, Jennifer Kean, Britta Moellers, Anthony A Ferrante, Steve Alam, Gregory O'Connor

[Mid-west Worm Meeting, 2002]

Autofluorescence background, the natural fluorescence from biological materials, reduces sensitivity of detection of localized fluorescence in multicellular organisms. For example in the case of a transgenic C. elegans that expresses GFP in two cells, that fluorescence can be masked by the autofluorescence of the remaining 1000 somatic cells. We have solved this problem with an add-on to our COPAS analysis and sorter technology that allows the instrument to measure and store the pattern of fluorescence along the length of the animal. That information was used to restrict fluorescence analysis to the region of the animal that contained the fluorescent cells thus improving the signal to autofluorescence ratio more than 20-fold. We used the Profiler system to perform a genetic screen in which we isolated mutants of PY1089 (kindly provided by Piali Sengupta), a transgenic C. elegans that expresses GFP in the AWB sensory neurons under control of the str-1 promoter. Proper expression of the odorant receptor, STR-1, in the AWB sensory neurons is required for appropriate chemotactic response to the repulsive odorant 2-nonanone. Without the Profiler technology the COPAS system was unable to distinguish PY1089 from N2 wild type animals, and it could not be used in a genetic screen for mutants with reduced GFP expression. With the addition of Profiler we were able to clearly distinguish PY1089 from N2 and were further able to sort animals that had either reduced or increased GFP expression in the sensory neurons. F2 progeny of mutagenized PY1089 were analyzed and sorted using the COPAS BIOSORT with prototype Profiler hardware. First, 100 animals were run and their peak fluorescence intensities were determined. That information was used to set a range of normal peak heights in the Profiler. The Profiler was instructed to dispense those animals whose peak heights were outside of the normal range, either higher or lower, one per well into 96-well plates. Mutants were confirmed by regrowing the sorted animals and running them through the Profiler a second time. Approximately 50,000 animals from 8 separate pools were screened and 1,000 were dispensed to wells in the first pass. We have retested 109 lines from the sorted animals to date. We have isolated 5 independent mutant lines with decreased GFP expression, 5 independent lines with increased GFP expression and 2 lines with a mix of increased, wild-type, and decreased GFP expression. So far, we have tested 5 mutant lines for their chemotactic response to 2-nonanone. Of these one line has demonstrated defective chemotaxis