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Genetics, Vol. 167, 1677-1687, August 2004, Copyright © 2004
doi:10.1534/genetics.103.024786

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A Network of Stimulatory and Inhibitory G{alpha}-Subunits Regulates Olfaction in Caenorhabditis elegans

Hannes Lans, Suzanne Rademakers and Gert Jansen1

MGC Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus Medical Center, 3000 DR Rotterdam, The Netherlands

1 Corresponding author: MGC Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands.
E-mail: g.jansen{at}erasmusmc.nl

Manuscript received November 20, 2003. Accepted for publication April 26, 2004.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
The two pairs of sensory neurons of C. elegans, AWA and AWC, that mediate odorant attraction, express six G{alpha}-subunits, suggesting that olfaction is regulated by a complex signaling network. Here, we describe the cellular localization and functions of the six olfactory G{alpha}-subunits: GPA-2, GPA-3, GPA-5, GPA-6, GPA-13, and ODR-3. All except GPA-6 localize to sensory cilia, suggesting a direct role in sensory transduction. GPA-2, GPA-3, GPA-5, and GPA-6 are also present in cell bodies and axons and GPA-5 specifically localizes to synaptic sites. Analysis of animals with single- to sixfold loss-of-function mutations shows that olfaction involves a balance between multiple stimulatory and inhibitory signals. ODR-3 constitutes the main stimulatory signal and is sufficient for the detection of odorants. GPA-3 forms a second stimulatory signal in the AWA and AWC neurons, also sufficient for odorant detection. In AWA, signaling is suppressed by GPA-5. In AWC, GPA-2 and GPA-13 negatively and positively regulate signaling, respectively. Finally, we show that only ODR-3 plays a role in cilia morphogenesis. Defects in this process are, however, independent of olfactory behavior. Our findings reveal the existence of a complex signaling network that controls odorant detection by C. elegans.

HETEROTRIMERIC G-proteins transduce diverse signals, varying from intercellular mediators to environmental stimuli (HAMM 1998). Activation of the G-protein complex, consisting of an {alpha}-, a ß-, and a {gamma}-subunit, results in exchange of GDP for GTP and release of the GTP-bound G{alpha}- and the Gß{gamma}-dimer. Both entities can activate effector molecules. In many species, ranging from yeast to mammals, multiple G{alpha}-, ß-, and {gamma}-genes, which may function simultaneously in the same cells, have been identified. It is often unclear how many and which G-protein pathways can be activated by a certain signal. Parallel activation of multiple pathways implies that specificity and cross-talk must be precisely regulated.

The olfactory system of Caenorhabditis elegans is adequately suited for studying G-protein signaling and its specificity. Five pairs of neurons, AWA, AWB, AWC, ASH, and ADL, are involved in olfaction, but only two of these, the AWA and AWC cells, detect attractive odorants (BARGMANN et al. 1993; TROEMEL et al. 1995, 1997). Both AWA and AWC neurons express many G-protein-coupled receptors (GPCRs) and at least three G{alpha}-subunits (TROEMEL et al. 1995; ZWAAL et al. 1997; ROAYAIE et al. 1998; JANSEN et al. 1999). Using these four cells, C. elegans can detect and discriminate many odorants (BARGMANN et al. 1993) and adapt to an odorant while remaining responsive to another (COLBERT and BARGMANN 1995). Part of the odorant specificity arises from the ability of the AWA and AWC cells to detect different odorants (BARGMANN et al. 1993). A functional asymmetry between the left and the right AWC cell provides a further means to discriminate between odorants (WES and BARGMANN 2001). Still, even functional differences between all four AWA and AWC cells cannot account for all observed odorant discrimination (WES and BARGMANN 2001), suggesting that additional intracellular mechanisms exist that establish odorant specificity.

Genetic screens have identified several genes involved in odorant detection (Table 1). Surprisingly, signal transduction in the AWA and AWC neurons is not identical. In both cell types, binding of an odorant to a GPCR is thought to activate the G{alpha}-protein ODR-3 (ROAYAIE et al. 1998). In the AWA cells, ODR-3 probably activates a TRPV channel consisting of the subunits OSM-9, OCR-1, and/or OCR-2 (COLBERT et al. 1997; TOBIN et al. 2002). In the AWC cells, ODR-3 induces an increase in intracellular cGMP, mediated by the guanylyl cyclases ODR-1 and DAF-11, leading to opening of the cyclic nucleotide-gated channel TAX-2/TAX-4 (COBURN and BARGMANN 1996; KOMATSU et al. 1996; BIRNBY et al. 2000; L'ETOILE and BARGMANN 2000). In addition to ODR-3, the G{alpha}-proteins GPA-2 and GPA-5 also are involved in regulating odorant responses (ROAYAIE et al. 1998; JANSEN et al. 1999). However, their exact function remains unclear. The functions of other olfactory G{alpha}-subunits have not been determined.


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  		TABLE 1 Signaling molecules involved in odorant detection by AWA and AWC

 
Previously, we have shown that, in addition to ODR-3 and GPA-2, the G{alpha}-proteins GPA-3, GPA-5, GPA-6, and GPA-13 function in the AWA and AWC neurons (JANSEN et al. 1999). In this study, we report a further characterization of all six olfactory G{alpha}-genes. Using antibodies, we show that they all, except GPA-6, localize to the ciliated endings of the sensory neurons. GPA-2, -3, -5, and -6 are also localized to the cell bodies and axons, GPA-5 is enriched at synaptic sites. Next, we show that, in the AWA neurons, ODR-3 and GPA-3 stimulate olfaction, whereas GPA-5 suppresses signaling. In the AWC neurons, olfaction is regulated by a balance between the stimulatory G{alpha}-subunits ODR-3, GPA-3, and GPA-13 and the inhibitory G{alpha} GPA-2. Finally, we show that only ODR-3 is involved in cilia morphogenesis. Although odr-3 mutants have flattened AWC cilia, this does not affect olfaction. Our analysis suggests that odorant perception requires a precisely balanced signaling network, consisting of both stimulatory and inhibitory signaling routes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Strains and plasmids:
Nematodes were grown at 20° or 25° on Escherichia coli strain OP50 using standard methods (BRENNER 1974). Wild-type animals were C. elegans variety Bristol, strain N2. Alleles used in this study were gpa-2(pk16) and gpa-3(pk35) (ZWAAL et al. 1997); gpa-3XS(pkIs508), gpa-5(pk376), gpa-5(pk377), gpa-5XS(pkIs379), gpa-6(pk480), gpa-6::GFP(pkIs583), gpa-6XS(pkIs519), and gpa-13(pk1270) (JANSEN et al. 1999); odr-1(n1936) (L'ETOILE and BARGMANN 2000); odr-3(n1605) (ROAYAIE et al. 1998); odr-7(ky4) (SENGUPTA et al. 1994); and str-2::GFP(kyIs140) (TROEMEL et al. 1999). Injection of transgenes, at 5–50 ng/µl, was performed according to standard methods (MELLO et al. 1991). Extrachromosomal transgenes were gpa-5::snb-1::GFP, gpa-6::GFP (JANSEN et al. 1999), gpa-13::GFP (JANSEN et al. 1999), and gpc-1::GFP (JANSEN et al. 2002). gpa-5::snb-1::GFP was created by fusing a 1385-bp NaeI-EcoRI snb-1::GFP fragment from pSB120 (a gift from M. Nonet; NONET 1999) to a 3724-bp PstI-SmaI fragment of the gpa-5 promoter in vector pPD95.79 (a gift from A. Fire).

Chemotaxis assays and statistical analysis:
Olfactory chemotaxis was tested at least four times on two separate days, as described (BARGMANN et al. 1993). Odorants used were diacetyl, pyrazine, benzaldehyde, 2,3-pentanedione, isoamyl alcohol, 2,4,5-trimethylthiazole, and butanone (Sigma Chemie, Acros Organics, and Fluka Chemie). A chemotaxis index was calculated as the number of animals at the odorant minus the number of animals at the control, divided by the total number of animals. The data obtained were analyzed using a one-way ANOVA test.

Immunofluorescence:
Animals were permeabilized, fixed, and stained according to standard methods (FINNEY and RUVKUN 1990). Polyclonal antibodies were generated by collecting serum from rabbits immunized with full-length, E. coli-produced, SDS-PAGE-purified G{alpha}-proteins fused to glutathione S-transferase. Staining was performed using dilutions of crude sera, or, if necessary, sera that were first affinity purified or precleared with acetone-fixed mutant animals. Secondary antibodies were goat-anti-rabbit Alexa-594-conjugated (Molecular Probes, Eugene, OR). Specificity of all antibodies was confirmed by the absence of immunoreactivity in G{alpha} null mutants.

Microscopy:
Antibody staining and cilia morphology were examined using a Leica DMRBE microscope, equipped with a Hamamatsu C4880 camera (Figure 1) and a Zeiss Axiovert 200, equipped with a Hamamatsu ORCA-ER camera (Figure 4). Individual cells were identified by using a combination of their position and morphology (WHITE et al. 1986) and the gpc-1::GFP construct (JANSEN et al. 2002). AWA and AWC cilia were identified by using gpa-6::GFP and gpa-13::GFP (JANSEN et al. 1999) constructs, respectively. While observing cells and cilia, we did not observe obvious defects in axon morphology or outgrowth in G{alpha}-mutants.



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  		FIGURE 1.— Intracellular localization of olfactory G{alpha}-subunits. In adult C. elegans (A), the olfactory G{alpha}-subunits locate to specific compartments of the amphid neurons. (B) The location and structures (dd, dendrite; cb, cell body; ax, axon) of the amphid neurons. GPA-2 (C) localizes to cilia (arrow) and cell bodies (arrowheads) of amphid neurons. GPA-13 (D) localizes to amphid cilia (arrow). GPA-5 (red) can be observed in the cilia (arrow), axons (open arrow), and cell bodies (arrowhead) of the AWA cells (E) and is localized nearby snb-1::GFP (green) in the axons (F, open arrows). GPA-6, in gpa-6XS animals, was not detected in the cilia, but in the dendrites (arrows), cell bodies (arrowhead), and axons (open arrow) of four pairs of amphid cells (G). GPA-3 localizes to the cilia of many amphid cells (H). GPA-3 staining in the rod-like cilia is clearly visible (arrow), surrounded by the more fuzzy wing-shaped AWA and AWC cilia staining (arrowheads). Bars: C, D, E, G, and H, 20 µm; F, 5 µm. (A–H) x400.

 


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  		FIGURE 4.— ODR-3 plays a role in cilia morphogenesis. (A–D) gpa-6::GFP expression in the AWA cilia. (E–H) gpa-13::GFP in the AWC and ADF/ASH cilia. The branched, filamentous structure of the AWA cilia is apparent from A–D. Because the structure of the AWC cilia is less apparent in E–H, a schematic of the cilia of AWC (blue) and ADF and ASH (red) is provided. The AWA cilia of wild type (A), odr-3 (B), gpa-3 odr-3 (C), and gpa-2 gpa-3 gpa-5 gpa-6 gpa-13 odr-3 sixfold mutants (D) are all extensively branched, despite olfactory differences. The AWA cilia of gpa-2 gpa-3 gpa-5 gpa-6 gpa-13 odr-3 mutants were difficult to visualize properly, because gpa-6::GFP is poorly expressed in these mutants. The AWC cilia of odr-3 (F) and gpa-2 gpa-3 gpa-5 gpa-6 gpa-13 odr-3 sixfold mutants (H) are less well differentiated than the AWC cilia of wild-type (E) and gpa-2 gpa-3 gpa-5 gpa-6 gpa-13 animals (G). For odr-3 cilia, see also ROAYAIE et al. (1998). Left is anterior. Bars, 5 µm. (A–H) x630.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Six G{alpha}-subunits localize to the cilia and axons of the AWA and AWC neurons:
Of the 21 G{alpha}-genes of C. elegans, 14 are specifically expressed in the amphid sensory neurons (ZWAAL et al. 1997; ROAYAIE et al. 1998; JANSEN et al. 1999; CUPPEN et al. 2003). We focused on G{alpha}-function in two pairs of amphid neurons, AWA and AWC. Each pair senses a different set of odorants (BARGMANN et al. 1993) and expresses at least three G{alpha}-genes. The AWC cells express gpa-2, gpa-13, and odr-3, while the AWA cells express gpa-5, gpa-6, and odr-3, as visualized using LacZ and GFP fusion constructs (ZWAAL et al. 1997; ROAYAIE et al. 1998; JANSEN et al. 1999). To determine the subcellular localization of the G{alpha}-subunits, we generated polyclonal antibodies and immunostained nematodes. The overall expression patterns of the G{alpha}-subunits, as observed with the antibodies, correlated well with the described expression patterns (Table 2).


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  		TABLE 2 G{alpha}-expression patterns and intracellular localization

 
The AWA and AWC cells each have an axon innervating the nerve ring, contacting other neurons, and a dendrite ending in elaborate cilia structures at the tip of the nose (WARD et al. 1975; WARE et al. 1975; WHITE et al. 1986). The AWA and AWC cilia can be recognized due to their widespread, wing-like morphology (see Figure 4, A and E), in contrast to the single or double-rod-like cilia of most of the other amphid neurons. In agreement with previous data (ROAYAIE et al. 1998), anti-ODR-3 antibodies were detected in the cilia of amphid neurons, while only faint staining of the dendrites and cell bodies was observed (Table 2). At high magnification, wing-shaped cilia, probably of the AWA, AWB, and AWC neurons, and the rod-like cilia of other amphid neurons, probably ASH and ADF, could be discerned (results not shown). In addition, our anti-ODR-3 serum also stained the cilia of one or both of the phasmid neurons PHA and PHB (Table 2).

Staining of the amphid cilia was also observed with anti-GPA-2, GPA-13, and GPA-5 antibodies (Figure 1, C–E). Anti-GPA-2 antibodies stained many cilia, cell bodies, and axons in the head (Figure 1C; results not shown). Two cell bodies in the anterior ganglion, two pharyngeal muscle cells, and seven cell bodies in the lateral and ventral ganglia showed staining, indicating that GPA-2 is expressed in more cells than initially identified (ZWAAL et al. 1997; Table 2). Importantly, in addition to rod-like cilia, the typical wing shape of the AWC cilia could be distinguished at high magnification (results not shown). This confirms that GPA-2 is expressed in the AWC cells. We found no indications of GPA-2 expression in the AWA cells.

Anti-GPA-13 antibodies stained the cilia of amphid and phasmid neurons (Figure 1D; Table 2). At high magnification, the AWC cilia and two slender cilia that probably belong to the ASH and ADF neurons could be observed (JANSEN et al. 1999; results not shown).

Anti-GPA-5 antibodies were detected not only in the cilia, but also in the cell bodies and axons of the AWA neurons (Figure 1, E and F). Often, although faintly, staining was observed in the ADL cell bodies, but not in the ASI cells (JANSEN et al. 1999; Table 2). A punctate staining pattern of the axon with anti-GPA-5 antibodies suggested that GPA-5 might be located at the synaptic sites of the AWA axons. Therefore, we generated animals that expressed a snb-1::GFP fusion construct specifically in AWA. Due to its fusion with SNB-1 synaptobrevin, green fluorescent protein (GFP) localizes to synaptic vesicles in these animals (NONET 1999). Staining with anti-GPA-5 serum showed a punctate localization of GPA-5 close to SNB-1::GFP (Figure 1F), suggesting that GPA-5 might function at the periactive zones of the AWA synapses.

The only G{alpha}-subunit that was not detected in the amphid cilia was GPA-6. In wild-type animals, no GPA-6 immunoreactivity could be detected. However, in animals overexpressing GPA-6, the dendrites, cell bodies, and axons of four pairs of amphid neurons, but not the cilia, showed staining (Figure 1G; Table 2). The positions of the cell bodies and colocalization experiments suggested that these cells are AWA, AWB, ADL, and ASH. No staining was observed in the ASI cells (JANSEN et al. 1999). The two pairs of phasmid neurons, PHA and PHB, and a third unidentified cell, posterior to these neurons, were also visible.

Previously, we found impaired odorant responses in nematodes overexpressing constitutively active GPA-3 (GPA-3QL; JANSEN et al. 1999). However, gpa-3::lacZ and GFP expression could not be detected in the AWA or AWC cells (ZWAAL et al. 1997; results not shown). We found anti-GPA-3 antibody staining in the cilia, cell bodies, and axons of many amphid cells (Figure 1H) and the two phasmid cells PHA and PHB (Table 2). The amphid cell bodies were faintly visible, but the typical wing shape of the AWC cilia could be recognized. Application of anti-GPA-3 antibodies to gpa-6::GFP animals showed GPA-3 colocalization with GPA-6::GFP in the AWA cilia and cell bodies (results not shown).

Taken together, our results suggest that ODR-3, GPA-2, GPA-3, GPA-5, and GPA-13 may be directly involved in chemosensory signaling in the amphid cilia, unlike GPA-6, and that GPA-5 may also function at the synapse.

ODR-3 and GPA-3 act redundantly in olfactory signaling:
To determine whether the G{alpha}-subunits are involved in odorant detection, we tested animals with loss-of-function (l.o.f.) mutations in one or more G{alpha}-subunits (ZWAAL et al. 1997; ROAYAIE et al. 1998; JANSEN et al. 1999) in odorant chemotaxis assays. First, the optimal chemotaxis conditions in our laboratory were determined. We obtained strong, reproducible chemoattraction with 1:10 and 1:100 dilutions of benzaldehyde, butanone, and isoamyl alcohol, whereas diacetyl, 2,4,5-trimethylthiazole and 2,3-pentanedione gave reliable results at dilutions ranging from 1:10 to 1:1000. Optimal pyrazine chemotaxis was achieved with 10 and 100 mg/ml concentrations. Low odorant concentrations are specifically detected by either the AWA or the AWC cells and higher concentrations by both (BARGMANN et al. 1993; CHOU et al. 2001). Therefore, we determined the cellular specificity of the odorant response, using odr-7 and odr-1 animals, which are defective in AWA and AWC olfaction, respectively (BARGMANN et al. 1993; SENGUPTA et al. 1994). Chemotaxis to butanone and isoamyl alcohol almost completely depended on AWC, whereas only low concentrations of benzaldehyde were specific to AWC. Low concentrations of diacetyl and pyrazine were specifically detected by the AWA cells, but high concentrations involved both AWA and AWC. The responses to 2,4,5-trimethylthiazole and 2,3-pentanedione seemed not specific to the AWA or AWC cells at the concentrations that gave strong, reproducible results (Table 3 and results not shown).


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  		TABLE 3 Cellular specificity of odorant detection

 
We tested the odr-3(n1605) l.o.f. mutant and confirmed that odr-3 is necessary for the detection of all odorants, at all concentrations tested (ROAYAIE et al. 1998; Figure 2, A–C; results not shown). In our assays, the response to butanone and low concentrations of diacetyl and benzaldehyde seemed to depend solely on odr-3. In contrast, the response to 2,3-pentanedione was only slightly affected. Other odorants gave intermediate phenotypes.



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  		FIGURE 2.— ODR-3 and GPA-3 form the major stimulatory signals in odorant detection. Chemotaxis of odr-3 animals and gpa-2 gpa-3 gpa-5 gpa-6 gpa-13 animals (shown as gpa-2 3 5 6 13) shows that ODR-3 is necessary and sufficient for chemotaxis to all odorants (see also ROAYAIE et al. 1998). GPA-3 is redundant to ODR-3 and is sufficient for chemotaxis at almost wild-type levels, as is evident from chemotaxis of gpa-3 animals, gpa-3 odr-3 double mutants, and gpa-2 gpa-5 gpa-6 gpa-13 odr-3 mutants (shown as gpa-2 5 6 13 odr-3). Mutants that lack all six olfactory G{alpha}-subunits (shown as "all") display no odorant attraction. Shown are the responses to odorants sensed primarily by AWA [A, 10 mg/ml pyrazine (pyr) and 1:1000 diacetyl (dia)], by AWA and AWC [B, 1:1000 2,4,5-trimethylthiazole (tmt) and 2,3-pentanedione (pent)], and by AWC [C, 1:100 benzaldehyde (benz), isoamyl alcohol (iaa), and butanone (but)]. D shows that chemotaxis to 50 mg/ml pyrazine of gpa-3 odr-3 mutants can be restored by introducing a gpa-3 transgene (shown as gpa-3 odr-3 gpa-3XS). Error bars denote standard error of the mean. Significant differences of odr-3 and gpa-3 animals compared to wild type (*, P ≤ 0.01), gpa-3 odr-3 compared to odr-3 animals (+, P ≤ 0.01; ++, P ≤ 0.05), and gpa-2 gpa-3 gpa-5 gpa-6 gpa-13 and gpa-2 gpa-5 gpa-6 gpa-13 odr-3 animals compared to gpa-2 gpa-3 gpa-5 gpa-6 gpa-13 odr-3 mutants (°, P ≤ 0.01; °°, P ≤ 0.05) are shown. In D, # denotes significant difference from gpa-3 odr-3 (P ≤ 0.01).

 
To identify the G{alpha} responsible for the residual response of odr-3 animals, we also tested other G{alpha} l.o.f. mutants, but observed no or only very mild effects (results not shown). This suggested that ODR-3 alone is sufficient for odorant detection. To test this, we generated animals with mutations in all six G{alpha}-subunits except odr-3. These gpa-2 gpa-3 gpa-5 gpa-6 gpa-13 mutants showed wild-type levels of chemotaxis to all odorants at all concentrations tested (Figure 2, A–C; results not shown), validating the idea that ODR-3 constitutes the major signaling pathway in AWA and in AWC. Additional loss of odr-3 abrogated chemotaxis to all odorants, except to pyrazine and high concentrations of 2,4,5-trimethylthiazole (Figure 2, A–C; results not shown). These odorants may also be sensed by other cells (BARGMANN et al. 1993).

Subsequently, to identify the G{alpha} mediating the residual response in odr-3 animals, double mutants between odr-3 and gpa-2, gpa-3, gpa-5, gpa-6, or gpa-13 were tested. Chemotaxis to all odorants was completely abolished in gpa-3 odr-3 mutants, at all concentrations tested (Figure 2, A–C; results not shown), indicating that GPA-3 also has a stimulatory role in odorant detection, redundant to ODR-3. This phenotype could be rescued by the introduction of a gpa-3 transgene (Figure 2D). To confirm that GPA-3 is functionally redundant to ODR-3, animals were generated with mutations in all six G{alpha}-subunits except gpa-3. Surprisingly, these gpa-2 gpa-5 gpa-6 gpa-13 odr-3 animals showed strong chemotaxis to all odorants, except to butanone (Figure 2, A–C; results not shown). Especially, chemotaxis to AWA-detected odorants was very strong. These results indicate that GPA-3 also is sufficient for detection of most odorants.

GPA-2, GPA-5, and GPA-13 modulate signaling via ODR-3 and GPA-3:
Previously, we have shown that GPA-5 negatively influences the response to 2,4,5-trimethylthiazole (JANSEN et al. 1999; Figure 3, A–C), suggesting that GPA-5 might have an inhibitory function. Further analysis of the olfactory response of gpa-5 odr-3 mutants showed that loss of GPA-5 suppressed the chemotaxis defect of odr-3 animals for all odorants detected by the AWA cells (Figure 3, A and B; results not shown). Suppression was strongest when using high odorant concentrations. The impaired chemotaxis of odr-3 animals to isoamyl alcohol, butanone, and low concentrations of benzaldehyde could not be suppressed by loss of GPA-5, in agreement with the fact that these odorants are detected by AWC, in which gpa-5 is not expressed (Figure 3C; results not shown). To confirm that GPA-5 caused the suppression of the odr-3 phenotype, we tested an independent gpa-5 l.o.f. allele (pk377), which gave similar results (results not shown).



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  		FIGURE 3.— GPA-5 and GPA-2 negatively regulate chemotaxis to most odorants, and GPA-13 has a stimulatory function. Chemotaxis of animals that lack ODR-3 and GPA-5 (gpa-5 odr-3) or GPA-2 (gpa-2 odr-3) reveals that GPA-5 and GPA-2 inhibit odorant detection in odr-3 animals. Furthermore, GPA-13 has a stimulatory function, which becomes apparent when gpa-2 is inactivated in an odr-3 background (compare gpa-2 odr-3 with gpa-2 gpa-13 odr-3). Shown are the responses to high concentrations of odorants sensed primarily by AWA [A, 100 mg/ml pyrazine (pyr) and 1:10 diacetyl (dia)], by AWA and AWC [B, 1:10 2,4,5-trimethylthiazole (tmt) and 2,3-pentanedione (pent)], and by AWC (C, 1:10 benzaldehyde (benz), isoamyl alcohol (iaa), and butanone (but)]. Low concentrations of diacetyl (1:1000, A) are also shown. D shows that GPA-13 is specifically involved in 2,3-pentanedione (1:1000 diluted; pent) signaling. Error bars denote standard error of the mean. Significant differences of gpa-5 odr-3, gpa-2 odr-3, and gpa-13 odr-3 animals compared to odr-3 animals (*, P ≤ 0.01; **, P ≤ 0.05), gpa-2 gpa-13 odr-3 compared to gpa-2 odr-3 animals (+, P ≤ 0.01; ++, P ≤ 0.05).

 
Next, we tested chemotaxis of gpa-2 odr-3 animals. Previously, a stimulatory function for GPA-2 was found in butanone perception (ROAYAIE et al. 1998). We found that GPA-2 negatively affects odorant detection, since gpa-2 odr-3 animals showed improved odorant responses as compared to odr-3 animals (Figure 3, A–C; results not shown). Like GPA-5, the inhibitory function of GPA-2 was clearest when using high odorant concentrations. Only isoamyl alcohol and butanone responses seemed not affected. Since the butanone response was completely abolished in odr-3 animals, a redundant stimulatory function for GPA-2 in butanone detection could not be analyzed (ROAYAIE et al. 1998).

Chemotaxis of gpa-6 odr-3 and gpa-13 odr-3 mutants also was tested. A function for GPA-6 in odorant detection could not be found, which is in agreement with its absence from the cilia. The behavior of gpa-13 odr-3 animals was indistinguishable from that of odr-3 animals, except for the response to low concentrations of 2,3-pentanedione (Figure 3D). In addition to this phenotype, we observed that inactivation of gpa-13 in gpa-2 odr-3 or gpa-5 odr-3 animals significantly reduced chemotaxis responses (Figure 3; results not shown), suggesting a minor stimulatory role for GPA-13 in olfactory signaling.

Taken together, our results confirm that ODR-3 constitutes the main stimulatory olfactory signaling pathway. Furthermore, our findings suggest that the olfactory defect of odr-3 mutants, for most odorants, is caused by the inhibitory action of GPA-2 and GPA-5. GPA-3 can fully substitute for ODR-3, but only when the function of GPA-2 or GPA-5 is lost and with the aid of GPA-13 or when all five other G{alpha}-subunits have been inactivated. Our data suggest that the olfactory response in C. elegans is regulated by a complex signaling network, which is activated upon odorant stimulation and may be necessary to obtain a precisely tuned response.

Olfactory defects are not caused by altered cilia morphology:
The odorant chemotaxis defects of G{alpha}-subunit mutants could be the result of developmental or structural defects of the sensory neurons. Such defects have been described for gpa-3 and odr-3 animals. The amphid neurons of gpa-3QL animals have lost the ability to take up fluorescent dyes (ZWAAL et al. 1997), probably because of structural defects of the cilia (J. BURGHOORN and G. JANSEN, unpublished results). Furthermore, the level of odr-3 expression controls AWA and AWC cilia morphology (ROAYAIE et al. 1998). Low levels of ODR-3 define flattened, filamentous cilia, but this is probably not the cause of the olfactory defects of odr-3 mutants. To rule out the possibility that altered cilia morphology causes the olfactory differences seen in G{alpha}-mutants, we analyzed the AWA and AWC cilia of these mutants in more detail. For this purpose, we used the G{alpha}-specific antibodies and the AWA-expressed gpa-6::GFP and AWC-expressed str-2::GFP and gpa-13::GFP transgenes.

The AWA cilia have a branched, filamentous shape, while the AWC cilia have a wide, wing-like structure (WARD et al. 1975; Figure 4, A and E). Unlike the majority of the amphid cilia, these cilia are not exposed to the environment (WARD et al. 1975; WARE et al. 1975).

First, single mutant animals were analyzed. All mutants, except odr-3 animals, had normal axon, dendrite, and cilia structures (Figure 4, B and F; results not shown). As previously described (ROAYAIE et al. 1998), the AWC cilia of odr-3 animals were flatter and less well differentiated, whereas the AWA cilia appeared normal. Next, gpa-3 odr-3 double mutants were analyzed. Although these mutants are more severely defective than odr-3 animals, their AWA and AWC cilia resembled those of odr-3 animals (Figure 4C; results not shown). Likewise, the cilia of animals with combinations of l.o.f. mutations in gpa-2, gpa-5, gpa-6, and gpa-13, some of which chemotax better than odr-3 animals (Figures 2 and 3), were comparable to odr-3 mutants (results not shown). These observations suggest that altered cilia morphology, as observed in odr-3 animals, does not cause the apparent olfactory defects or that impaired sensory signaling causes cilia degradation.

Next, five- and sixfold G{alpha}-mutants were examined. The AWA cilia appeared as wild type in animals that expressed only gpa-3, gpa-5, gpa-6, or odr-3, although these mutants show varying chemotaxis (Figure 2; results not shown). Also, animals that had lost all six G{alpha}-subunits showed wild-type AWA cilia (Figure 4D). With regard to the AWC cilia, nematodes that expressed only gpa-2, gpa-3, or gpa-13 and animals with l.o.f. mutations in all six olfactory G{alpha}-subunits had flattened AWC cilia, similar to odr-3 animals (Figure 4H; results not shown). In contrast, animals that expressed only odr-3, i.e., gpa-2 gpa-3 gpa-5 gpa-6 gpa-13 mutants, had wild-type AWC cilia (Figure 4G). These results indicate that, of the four AWC-expressed G{alpha}-subunits, only ODR-3 is necessary to establish normal AWC cilia morphology. The other five olfactory G{alpha}-subunits are not involved in AWA or AWC cilia development.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Olfaction using four cells requires signaling via five G{alpha}-subunits:
Our results show that olfactory signaling in C. elegans is more complex than initially realized. Five G{alpha}-subunits regulate the response of C. elegans to attractive odorants (Figure 5). ODR-3 constitutes the main stimulatory signal in the AWA and AWC cells. GPA-3 also provides a stimulatory signal in both neuron pairs. GPA-5 and GPA-2 have inhibitory functions in the AWA and the AWC cells, respectively, and GPA-13 has a minor stimulatory role in the AWC cells. Although the odorant concentrations used in this study are not all detected specifically by either AWA or AWC, it is highly likely that the observed regulation by G{alpha}-subunits occurs only in these cells. First of all, no other cells have been reported to be involved in odorant attraction in C. elegans. Second, GPA-5 is expressed only in AWA and faintly in ADL, whereas AWC is the only sensory amphid cell in which GPA-2 is expressed. Finally, AWA-specific expression of GPA-3 rescues AWA-mediated olfaction in a gpa-3 odr-3 background (H. LANS and G. JANSEN, unpublished results).



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  		FIGURE 5.— Model of G{alpha} signaling during odorant detection via the AWA and AWC cells. In AWA, both ODR-3 and GPA-3 can transduce the odorant signal to the TRPV channel OSM-9/OCR-1/OCR-2, but ODR-3 forms the major pathway. GPA-5, at an unknown effector, cross-interacts with this signal to inhibit signaling. GPA-5 might be activated by the same receptor as ODR-3 and GPA-3 or by another receptor. In AWC, mainly ODR-3, but also GPA-3, transduces the odorant signal to the guanylyl cyclases ODR-1 and DAF-11, leading to the opening of the TAX-2/TAX-4 ion channel. GPA-2 and GPA-13 inhibit and stimulate this signal, respectively.

 
We cannot exclude that other ubiquitously expressed G{alpha}-subunits, like goa-1 (MENDEL et al. 1995; SEGALAT et al. 1995), gsa-1 (KORSWAGEN et al. 1997), egl-30 (LACKNER et al. 1999), and gpa-7 (JANSEN et al. 1999), also function in the AWA and AWC cells. However, on the basis of the data presented in this study, it is not likely that one of these is required for olfactory signaling. It is striking that signaling in AWA and AWC, the only cells required for odorant attraction (BARGMANN et al. 1993), involves different G{alpha}-subunits. Interestingly, this correlates well with the existence of dissimilar downstream signaling pathways in the two neuron pairs (Table 1; Figure 5). In AWA, signaling is reminiscent of Drosophila phototransduction, due to its dependence on TRP channel proteins (COLBERT et al. 1997; HARDIE and RAGHU 2001; TOBIN et al. 2002). AWC signaling, on the other hand, resembles that in the mammalian main olfactory epithelium, where all signals converge on a cyclic nucleotide-gated channel (COBURN and BARGMANN 1996; KOMATSU et al. 1996; FIRESTEIN 2001). Signaling in this system also involves a separate modulatory pathway and multiple G{alpha}-proteins (JONES and REED 1989; BELLUSCIO et al. 1998; WEKESA and ANHOLT 1999; LUO et al. 2002; SPEHR et al. 2002).

Why would C. elegans need several G{alpha}-subunits with overlapping or antagonizing functions to modulate olfaction? We hypothesize that such a balanced signaling network is necessary to allow adjustment of the response to an odorant when odorant concentrations or other environmental conditions change. The inhibitory effects of GPA-2 and GPA-5 were most obvious when the animals were exposed to high odorant concentrations. In these circumstances, desensitization or adaptation mechanisms may become activated. Following prolonged exposure to high odorant concentrations, C. elegans shows a diminished response to that odorant, but not to other odorants (COLBERT and BARGMANN 1995). This process, which is called adaptation, involves the unknown adp-1 gene, the TRPV channel subunit OSM-9, and the cGMP-dependent protein kinase EGL-4 (COLBERT and BARGMANN 1995; L'ETOILE et al. 2002). Since odorant adaptation is triggered by calcium and cGMP levels, it seems likely that this response is regulated by stimulating and inhibiting G{alpha}-subunits. Furthermore, odorant-specific adaptation is modulated by other environmental cues, which are probably transduced by G-proteins. For example, the absence of food stimulates benzaldehyde adaptation, but not isoamyl alcohol adaptation (COLBERT and BARGMANN 1997). Similarly, the presence of food suppresses benzaldehyde adaptation (NUTTLEY et al. 2002). Although it is uncertain where the integration of signals like food availability and odorants occurs, multiple G-proteins within one cell might be essential to control the proper response of an animal to several simultaneous cues.

In addition, overlapping G-protein pathways could facilitate discrimination between odorants detected by the same neuron (WES and BARGMANN 2001). Our finding that two G{alpha}-subunits, ODR-3 and GPA-3, can mediate the detection of all odorants provides a basis for this idea. Inhibition of ODR-3 and GPA-3 signaling by GPA-2 and GPA-5 could also be a means for odorant discrimination. Both adaptation and discrimination assays could shed more light on the involvement of G-proteins in adaptation and discrimination.

Finally, another hint at the biological significance of the G-protein network could be provided by the finding that vulval induction by the Ras-mitogen-activated protein kinase (MAPK) pathway is negatively regulated by GPA-5 and the GPCR SRA-13, depending on food conditions (BATTU et al. 2003). This indicates that olfactory signaling not only serves to direct movement toward food, but also may regulate developmental processes.

Specificity of G-protein signaling pathways:
Our genetic analysis of G-protein function in olfactory signaling shows that the G{alpha}-subunits have specific functions, despite their shared localization in the cell. This raises the question of how specificity is organized and maintained.

First of all, specificity may be defined by specific interactions with receptors, Gß{gamma}-subunits, and effectors. It is likely that ODR-3 and GPA-3 are activated by the same receptors during odorant detection. GPA-2 and GPA-5 might inhibit signaling by competing for these receptors, but could also be activated by different receptors or in a receptor-independent fashion. Recently, GPA-5 and SRA-13 have been found to negatively regulate a Ras/MAPK pathway downstream of ODR-3 in the AWC cells (HIROTSU et al. 2000; BATTU et al. 2003). Although this could provide a mechanism through which ODR-3 and GPA-3 signaling is inhibited, it is unclear how GPA-5 could function in the AWC cells. Furthermore, it is striking that no effect of GPA-2 on the Ras/MAPK pathway has been observed (BATTU et al. 2003).

Another determinant is provided by the ß{gamma}-dimer that associates with the G{alpha}-subunits. C. elegans has two Gß- and two G{gamma}-subunits, GPB-1 and -2 and GPC-1 and -2 (VAN DER VOORN et al. 1990; ZWAAL et al. 1996; JANSEN et al. 1999). GPC-1 functions specifically in a subset of sensory cells, but not in AWA and AWC (JANSEN et al. 2002). Therefore, the ubiquitously expressed GPC-2 (JANSEN et al. 2002) is the likely candidate to interact with the olfactory G{alpha}-subunits. In AWA and AWC, GPC-2 may dimerize with either GPB-1 or GPB-2, which are both widely expressed (CHASE et al. 2001; VAN DER LINDEN et al. 2001). Alternatives to GPC-2, however, could be the G{gamma}-like domain containing RGS proteins EAT-16 and EGL-10, which can interact with GPB-2 (CHASE et al. 2001; ROBATZEK et al. 2001; VAN DER LINDEN et al. 2001). Thus, together there are four different Gß{gamma}(-like) partners. It is difficult to test which of these partners functions in vivo because both gpb-1 and gpc-2 mutations are lethal (ZWAAL et al. 1996; GOTTA and AHRINGER 2001).

A spatial separation of different pathways is a further means to confer specificity. Within the cilia, compartmentalization could separate the different G{alpha}-subunits, as was previously suggested to insulate cGMP signaling during odorant discrimination and adaptation (L'ETOILE and BARGMANN 2000; L'ETOILE et al. 2002).

A third method to provide specificity could be a temporal induction of G-protein signaling. For example, the translocation of the cGMP-dependent protein kinase EGL-4 to the nucleus of the AWC cells, following long-term adaptation to odorants (L'ETOILE et al. 2002), may induce the expression of genes necessary for adaptation that would otherwise interfere with olfactory signaling.

It is becoming increasingly clear that most signaling pathways are part of complex, intracellular signaling networks. Our characterization of the olfactory system of C. elegans provides us with the possibility of identifying the molecular mechanisms that regulate specificity in vivo.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
LITERATURE CITED
 
We thank Ronald Plasterk in whose lab this project was started. We acknowledge the assistance of Marieke van der Horst and Gert van Cappellen. We thank Cori Bargmann and the Caenorhabditis Genetics Center for strains and Andy Fire and Michael Nonet for plasmids. This work was supported by the Netherlands Organization for Scientific Research (NWO, grant ALW 805-48-009), the Royal Dutch Academy of Sciences (KNAW), and the Center for Biomedical Genetics, Rotterdam, The Netherlands.


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 DISCUSSION
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H. Lans and G. Jansen
Noncell- and Cell-Autonomous G-Protein-Signaling Converges With Ca2+/Mitogen-Activated Protein Kinase Signaling to Regulate str-2 Receptor Gene Expression in Caenorhabditis elegans
Genetics, July 1, 2006; 173(3): 1287 - 1299.
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