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MGC Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus MC, 3000 DR Rotterdam, The Netherlands
1 Corresponding author: Department of Cell Biology and Genetics, Erasmus MC, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands.
E-mail: g.jansen{at}erasmusmc.nl
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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subunits ODR-3, GPA-2, GPA-5, and GPA-6 and a Ca2+/MAPK pathway involving the Ca2+ channel subunit UNC-36, the CaMKII UNC-43, and the MAPKK kinase NSY-1 induce strong str-2 expression. Cell-specific rescue experiments suggest that ODR-3 and the Ca2+/MAPK genes function in the AWC neurons, but that GPA-5 and GPA-6 function in the AWA and ADL neurons, respectively. In Dauer larvae, the same network of genes promotes strong str-2 expression in the ASI neurons, but ODR-3 functions in AWB and ASH and GPA-6 in AWB. Our results reveal a complex signaling network, encompassing signals from multiple cells, that controls the level of receptor gene expression at different developmental stages.
The nematode Caenorhabditis elegans senses chemical cues in its environment using 11 bilateral pairs of chemosensory amphid neurons. Cell ablation experiments have shown that these cells are involved in the response to specific sensory cues (BARGMANN and MORI 1997). The AWA and AWC neurons detect attractive odorants, whereas the AWB, ASH, and ADL neurons detect repulsive odorants. The ASE, ADF, ASK, ASG, ASI, ADL, and ASH neurons respond to nonvolatile chemicals and the ADF, ASI, ASG, and ASJ neurons are involved in the response to Dauer pheromone.
The genome of C. elegans encodes 
800 functional receptor genes, many of which are specifically expressed in the amphid neurons (TROEMEL et al. 1995; BARGMANN 1998; ROBERTSON 1998; ROBERTSON 2000). Binding of a ligand to a receptor activates one or more heterotrimeric G proteins, consisting of G-, Gß-, and G
-subunits, leading to the activation of a cascade of effectors. The sensory neurons of C. elegans express 14 G-subunits, several of which have been shown to be involved in olfaction, pheromone perception, and taste (ZWAAL et al. 1997; ROAYAIE et al. 1998; JANSEN et al. 1999; HILLIARD et al. 2004; LANS et al. 2004; HUKEMA et al. 2006). For example, detection of attractive odorants involves at least 5 G-subunits (ROAYAIE et al. 1998; LANS et al. 2004). The G-subunit ODR-3 constitutes the major signaling route and is sufficient for odorant detection. The G-subunit GPA-3 is redundant to ODR-3 and also sufficient for most odorant signals. Signaling via ODR-3 and GPA-3 is inhibited by GPA-5 and GPA-2 in the AWA and AWC neurons, respectively, and stimulated by GPA-13 in the AWC neurons. The presence of multiple receptors and G proteins per cell probably enables C. elegans to respond to a wide variety of sensory cues, using only few cells.
Receptor expression is regulated by developmental and experience-based signals, allowing C. elegans to adjust behavior in changing environments. Receptor expression can vary from embryos to adults or from males to hermaphrodites (TROEMEL et al. 1995, 1999) and it can change upon entry into the Dauer larval stage (PECKOL et al. 2001). In addition, expression of the srd-1, str-2, and str-3 receptor genes in adult animals is repressed by Dauer pheromone and srd-1 expression is abolished in the absence of sensory signaling (PECKOL et al. 2001).
In well-fed adult animals, the candidate odorant receptor gene str-2 is strongly expressed in either the left or the right AWC neuron and weakly expressed in both ASI neurons (TROEMEL et al. 1999). In Dauer animals, str-2 is repressed in AWC and strongly expressed in both ASI neurons (PECKOL et al. 2001). Early in development, probably in the late embryo, a stochastic choice is made to repress str-2 expression in one of the two AWC neurons. This is accomplished by Ca2+ signaling via the N/P-type voltage-gated Ca2+ channel UNC-2/UNC-36 and the Ca2+/calmodulin-dependent protein kinase II (CaMKII) UNC-43, which activates a MAPK pathway consisting of the MAPKK kinase NSY-1 and the MAPK kinase SEK-1 (TROEMEL et al. 1999; SAGASTI et al. 2001; TANAKA-HINO et al. 2002). Also, the Ca2+-activated K+-channel NSY-3/SLO-1 and the TIR protein TIR-1 are involved (SAGASTI et al. 2001; DAVIES et al. 2003; CHUANG and BARGMANN 2005). Furthermore, mutations that disrupt axon guidance impair asymmetric str-2 expression and killing one AWC neuron in the embryo abolishes str-2 expression, suggesting that the asymmetry is initiated by lateral AWC axon contact (TROEMEL et al. 1999). Once asymmetry is established, str-2 expression is maintained by the activity of several olfactory signaling molecules, such as the guanylyl cyclases ODR-1 and DAF-11 and the cyclic nucleotide-gated channel TAX-2/TAX-4 (TROEMEL et al. 1999). The regulation of str-2 expression in the ASI neurons seems to involve different mechanisms, because no effects of unc-36 and tax-2/-4 mutations have been observed (PECKOL et al. 2001). ASI expression is suppressed by Dauer pheromone and is under the control of genes of the TGFß-signaling pathway, which also regulates Dauer development (PECKOL et al. 2001; NOLAN et al. 2002).
Since several lines of evidence suggest that receptor expression is regulated by sensory signaling, we studied the involvement of the olfactory G-subunits in regulating str-2 expression. We show that G proteins regulate str-2 gene expression, but do not affect the developmental choice to express str-2 asymmetrically. Four G-subunits, ODR-3, GPA-2, GPA-5, and GPA-6, together with Ca2+ signaling through UNC-36, UNC-43, and NSY-1, promote str-2 expression in the AWC and ASI neurons of young larvae, Dauer larvae, and adults. Interestingly, this regulation involves G-protein signaling in neurons other than AWC and ASI and does not seem to require sensory cues.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Transgenes and germline transformation:
To examine str-2 and odr-10 expression, the integrated alleles kyIs140[str-2::gfp] (TROEMEL et al. 1999) and kyIs37[odr-10::gfp] (SENGUPTA et al. 1996) were used. All other transgenes were generated by standard germline transformation (5–100 ng/µl; MELLO et al. 1991) and maintained extrachromosomally using the elt-2::gfp construct as the co-injection marker (FUKUSHIGE et al. 1999).
Promoters used were gpa-11 (ADL, ASH; JANSEN et al. 1999), gpa-13 (ADF, ASH, AWC; JANSEN et al. 1999), hsp-16.2 (a gift from A. Fire), odr-10 (AWA; SENGUPTA et al. 1996), sra-6 (ASH, ASI, PVQ, SPDm/SPVm; TROEMEL et al. 1995), srh-142 (ADF; SAGASTI et al. 1999), sro-1 (ADL, SIA; TROEMEL et al. 1995), and str-1 (AWB; TROEMEL et al. 1997). All expression patterns were confirmed by examining promoter::gfp fusion constructs (using pPD95.79, a gift from A. Fire) in wild-type and unc-36; odr-3; gpa-5 gpa-6 animals, except sro-1::gfp, which was observed in additional cells but not in other sensory neurons. To further confirm promoter specificity (Figure 4), immunofluorescence was performed as described (LANS et al. 2004). Although the levels of expression of the different promoters used for rescue varied, these levels did not correlate with rescue (results not shown). For example, gpa-6 constructs driven by either a relatively weak or a strong promoter, sro-1 and gpa-11, respectively, could rescue str-2 expression.
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Microscopy:
To examine str-2 expression, animals were allowed to lay eggs for 2–5 hr at 20° and then removed. Their offspring was picked at different developmental stages to score str-2::gfp levels (3–4 days for adults). str-2::gfp was scored and images in Figure 1 were acquired using a Leica Aristoplan microscope, equipped with a Sony DXC-9508 3CCD camera. Expression was scored as "strong" if GFP fluorescence was detected at x100 magnification, regardless of brightness, as "weak" if GFP was detected only at x400, and as "off" if GFP could not be detected at x400. Statistical analysis was performed using a two-sample test of proportion. Images in Figure 4 were acquired using a Zeiss LSM510 microscope.
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3 hr on plates containing 40 µl of pheromone, the minimum required to induce 80% wild-type Dauer formation. The plates were incubated at 25° for 3 days, after which str-2 expression was scored. |
RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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-subunit mutations:-subunits in its olfactory neurons AWA and AWC: GPA-2, GPA-3, GPA-5, GPA-6, GPA-13, and ODR-3 (ZWAAL et al. 1997; ROAYAIE et al. 1998; JANSEN et al. 1999; LANS et al. 2004). All, except GPA-6, play a role in odorant detection. To determine their involvement in regulating gene expression, we examined the asymmetric expression of str-2 in the AWC cells (Figure 1A) of several G loss-of-function mutants. For this purpose, the kyIs140[str-2::gfp] allele (TROEMEL et al. 1999) was introduced in gpa-2, gpa-3, gpa-5, gpa-6, gpa-13, and odr-3 mutants. No change in the asymmetry of str-2 expression was observed in these mutants, as was already found for odr-3 (results not shown; TROEMEL et al. 1999).
In the G-mutants, we observed only limited variation in str-2 expression levels (Table 1). This was unexpected, since mutations in the cGMP-signaling genes odr-1, daf-11, tax-2, and tax-4, which are likely activated through G-protein signaling, dramatically reduce str-2 expression (Table 1; TROEMEL et al. 1999). To test whether this discrepancy resulted from functional redundancy of the G-subunits, str-2 expression was examined in animals with mutations in two to six G-subunits. Surprisingly, in most mutants the strength of expression was close to normal and no effect on the asymmetry of str-2 expression was observed (Table 1; results not shown). Only odr-3; gpa-6 double mutants displayed a strong reduction, which was partially (gpa-3 or gpa-5) or completely (gpa-2 or gpa-13) restored by additional mutations in other G-subunits (Figure 1B; Table 1). Animals that had lost the function of all six G-subunits also displayed a clear reduction of str-2::gfp strength, but not as strong as that of odr-3; gpa-6 animals (Figure 1C; Table 1). These results show that the six olfactory G-subunits regulate str-2 expression in a redundant fashion, but do not influence the asymmetry of str-2 expression.
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-mutations on str-2 expression resemble the effects of mutations in the odr-1, daf-11, tax-2, and tax-4 genes. Mutations in these genes do not affect the decision to express str-2 asymmetrically, but affect maintenance of str-2 expression once asymmetry is established (TROEMEL et al. 1999). In tax-4 and odr-1 L1 larvae, str-2 is expressed strongly, but during later stages expression is reduced or even disappears (Table 1; TROEMEL et al. 1999). To determine whether the olfactory G-subunits function to maintain expression, we determined str-2::gfp strength in the L1 larvae of several G-mutants (Table 1; results not shown). In odr-3; gpa-6 larvae, we observed relatively higher str-2 expression than we observed in adults. However, these and gpa-2 gpa-3 gpa-13 odr-3; gpa-5 gpa-6 larvae still showed a reduction in str-2::gfp strength when compared to wild-type L1 larvae. These results suggest that the G-subunits function predominantly to maintain str-2 expression, but also contribute to the initiation of str-2 expression.
G proteins cooperate with Ca2+/MAPK signaling to regulate str-2 expression:
Early in development, the decision to express str-2 asymmetrically is executed by Ca2+ and MAPK signaling (TROEMEL et al. 1999; SAGASTI et al. 2001; TANAKA-HINO et al. 2002; CHUANG and BARGMANN 2005). We tested the involvement of G proteins by examining str-2 expression in animals with mutations in Ca2+/MAPK signaling genes and G-subunits. Mutations in the Ca2+ channel subunit unc-36 result in strong str-2 expression in both AWC neurons, indicating that in wild-type animals UNC-36 represses str-2 expression in one neuron (TROEMEL et al. 1999; Figure 1D). Additional mutations in G-subunits never changed this symmetric expression (results not shown), but, intriguingly, affected the strength of str-2 expression in unc-36 mutants (Figure 2A; results not shown). A mild reduction in str-2 expression was observed in unc-36; odr-3 and unc-36; odr-3; gpa-6 mutants. However, an unc-36 mutation, together with different combinations of three or four G-subunit mutations, including gpa-2, odr-3, gpa-5, or gpa-6, severely reduced expression (Figures 1E and 2A). Reduction of str-2 expression was strongest in unc-36; odr-3; gpa-5 gpa-6 animals, in which, at x400 magnification, almost no fluorescence could be observed (results not shown). Interestingly, expression was restored almost to wild-type levels when all six olfactory G-subunits were inactivated (Figure 1F). These results indicate that ODR-3, GPA-2, GPA-5, and GPA-6 cooperate with UNC-36 to promote str-2 expression, but that GPA-3 and GPA-13 inhibit str-2 expression.
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-subunits restored str-2 expression to wild-type levels. In nsy-1 mutants, loss of odr-3, gpa-5, and gpa-6 had no effect, but loss of gpa-2, odr-3, gpa-5, and gpa-6 severely reduced str-2 expression levels (Figure 2B; results not shown). Again, loss of all olfactory G-subunits restored str-2 expression levels. Unexpectedly, in tir-1 and nsy-3 animals, loss of gpa-2, odr-3, gpa-5, and gpa-6 did not result in a strong reduction of expression (Figure 2C), even though TIR-1 has been reported to be necessary to localize NSY-1 correctly to postsynaptic sites (CHUANG and BARGMANN 2005). sek-1 mutants already showed a mild reduction in str-2 expression levels, which was only very mildly enhanced by additional G-mutations (Figure 2C; results not shown). These data suggest that, parallel to part of the Ca2+/MAPK pathway, several G-protein-signaling pathways that regulate str-2 expression exist. Furthermore, these results reveal dual functions for unc-36, unc-43, and nsy-1 in the AWC neurons: suppression of str-2 expression in one AWC cell to establish str-2 asymmetry and promotion of str-2 expression together with G-protein signaling. To determine if the signaling network of G proteins and Ca2+/MAPK genes is required when str-2 expression is initiated or to maintain expression, str-2 expression was determined at different time points during development (Figure 2D). In unc-36; odr-3; gpa-5 gpa-6 L1 larvae, str-2 expression was weak, but increased considerably in successive larval stages. In adults, str-2 expression diminished again. In contrast, str-2 expression in the AWC neurons of odr-1 animals disappeared completely after the L1 stage. These results suggest that str-2 expression levels are controlled by the G-protein and Ca2+/MAPK-signaling network early during development and in adults. Other mechanisms might promote str-2 expression from the L2 to L4 stage.
str-2 expression does not require sensory cues:
Because str-2 expression maintenance requires genes that are essential for olfaction (COBURN and BARGMANN 1996; KOMATSU et al. 1996; TROEMEL et al. 1999; BIRNBY et al. 2000; L'ETOILE and BARGMANN 2000; this study), it was suggested that str-2 expression is regulated by sensory cues. To test this, we introduced str-2::gfp in osm-6 and dyf-8 mutants, which have impaired chemosensory responses due to structural defects of the cilia (PERKINS et al. 1986; STARICH et al. 1995). osm-6 mutants showed no chemotaxis to various salts or avoidance of various water-soluble repellents (CULOTTI and RUSSELL 1978; PERKINS et al. 1986; HILLIARD et al. 2004), but still showed a significant response to AWA- and AWC-sensed odorants, albeit strongly reduced (results not shown). dyf-8 animals, however, showed a completely disrupted response to odorants and salts (STARICH et al. 1995; results not shown). In addition, visualization of the sensory neurons using various gfp fusion constructs showed that the dendrites of the sensory neurons of dyf-8 animals are truncated (H. LANS, J. BURGHOORN and G. JANSEN, unpublished results). Surprisingly, osm-6 and dyf-8 animals showed wild-type levels of str-2 expression in the AWC neurons (Table 1). Also, unc-36; osm-6 double mutants showed almost wild-type str-2 expression levels (Table 1). These results indicate that expression of str-2 does not require sensory activity per se. Expression of str-2 in the ASI neurons has previously been shown to be unaffected by structural defects of the cilia (PECKOL et al. 2001).
AWC cell fate is unaltered in unc-36; odr-3; gpa-5 gpa-6 mutants:
Reduction of str-2 expression could be caused by general developmental or structural defects caused by loss of G-protein signaling. ODR-3 has a function in morphogenesis of the AWC cilia, but this is independent of sensory signaling (ROAYAIE et al. 1998; LANS et al. 2004). We observed no abnormalities of the axons or dendrites of unc-36; odr-3; gpa-5 gpa-6 mutants (results not shown). Also, the amphid neurons of these mutants showed normal uptake of fluorescent dyes, suggesting that their cilia are intact (results not shown; PERKINS et al. 1986). To test whether structural defects caused the reduction of str-2 expression, we tested if str-2 expression could be restored by reintroduction of ODR-3. Following heat shock of adult unc-36; odr-3; gpa-5 gpa-6 animals carrying an odr-3 gene under control of a heat-shock promoter, str-2 expression was significantly upregulated (Figure 2E). This shows that str-2 can be expressed immediately in response to an activating signal, suggesting that the AWC neurons develop correctly.
The function of a sensory neuron can be defined by the sensory signaling and structural genes that it expresses. Therefore, reduction of str-2 expression might be caused by partial or complete loss of AWC identity. This is the case for animals with mutations in the Otx homeodomain gene ceh-36, which have no odr-1 and str-2 expression in the AWC neurons and do not respond to AWC-sensed odorants (LANJUIN et al. 2003). To test if expression of additional AWC-specific genes was affected in unc-36; odr-3; gpa-5 gpa-6 animals, we introduced odr-1::gfp and gpa-13::gfp fusion constructs in these mutants (JANSEN et al. 1999; L'ETOILE and BARGMANN 2000). Both genes were strongly expressed in the AWC neurons, confirming that basal AWC neurons develop normally in unc-36; odr-3; gpa-5 gpa-6 animals (results not shown).
Next, we wondered whether GPA-5, GPA-6, ODR-3, and UNC-36 regulate not only the expression of str-2 but also that of other receptor genes. To test this, we examined the expression of odr-10, expressed in AWA (SENGUPTA et al. 1996); str-1, expressed in AWB (TROEMEL et al. 1997); srh-142, expressed in ADF (SAGASTI et al. 1999); sra-6, expressed in ASH, and sro-1; expressed in ADL (TROEMEL et al. 1995). All receptors showed comparable expression levels in wild-type animals and in unc-36; odr-3; gpa-5 gpa-6 animals in the appropriate cells (results not shown). These results suggest that the signaling network under study specifically regulates the expression of str-2.
ODR-3 and UNC-36 act in AWC, GPA-5 in AWA, and GPA-6 in ADL to regulate str-2 expression in AWC:
Previously, using gfp fusion constructs and antibodies, it was shown that GPA-5 and GPA-6 are not expressed in the AWC neurons, but in other sensory neurons (JANSEN et al. 1999; LANS et al. 2004). These observations, together with our data, imply that signals from other sensory neurons regulate str-2 expression in AWC. We decided to identify the neurons in which ODR-3, GPA-5, GPA-6, and UNC-36 function by using cell-specific rescue constructs. First, we generated gfp fusion constructs to confirm the cellular expression patterns of the promoters that we would use (see MATERIALS AND METHODS). All promoters used for cell-specific rescue showed expression in the correct sensory neurons in both wild-type and unc-36; odr-3; gpa-5 gpa-6 animals (results not shown).
ODR-3 is expressed in the amphid neurons AWA, AWB, AWC, ASH, and ADF (ROAYAIE et al. 1998). Introduction of the wild-type odr-3 gene in unc-36; odr-3; gpa-5 gpa-6 animals fully restored str-2 expression (Figure 3A; Table S1 at http://www.genetics.org/supplemental/). Full rescue was also obtained when ODR-3 was expressed under the gpa-13 promoter, which drives expression in AWC and in the ASH and ADF cells (JANSEN et al. 1999). These results are consistent with a function of ODR-3 in the AWC neurons. In agreement, specific expression of ODR-3 in the AWA, AWB, or ASH neurons, using the odr-10, str-1, and sra-6 promoter, did not or only very weakly restore str-2 expression (Figure 3A; Table S1). In addition, a weak restoration of str-2 expression was observed when ODR-3 was expressed in the ADF neurons, using the srh-142 promoter (Figure 3A; Table S1). Simultaneous expression of odr-3 in the ASH and ADF neurons, but not AWC, had no additional effect (results not shown). To rule out that the observed differences in restoration of str-2 expression resulted from differences in expression levels of the promoters rather than from differences in cell-specific expression, we introduced the odr-3 rescue constructs at different concentrations (5–100 ng/µl). We observed consistent rescue of a given construct at various concentrations (results not shown). To confirm correct ODR-3 expression in the AWC and ADF cells, we applied antibodies to visualize ODR-3. Animals expressing odr-3 under control of the gpa-13 promoter showed strong anti-ODR-3 staining in the cilia of the AWC neurons, which can be recognized by their wing-like morphology (Figure 4A). Weak staining in the cilia of the ASH and/or AFD neurons could be observed only occasionally, indicating that gpa-13::odr-3 is predominantly expressed in the AWC neurons. Animals expressing ODR-3 under control of the srh-142 promoter showed staining in the ADF cilia only (Figure 4B). These results suggest that ODR-3 functions predominantly in the AWC neurons and perhaps to some extent in the ADF neurons to regulate str-2 expression; however, we cannot exclude that it functions in some combination of the AWC and ADF or ASH neurons.
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GPA-6 is expressed in the amphid neurons AWA, AWB, ADL, and ASH, but not in the AWC neurons (JANSEN et al. 1999; LANS et al. 2004). str-2 expression could be restored using an integrated and an extrachromosomal gpa-6 transgene (Figure 3C; Table S3 at http://www.genetics.org/supplemental/). To find out in which neurons GPA-6 functions, GPA-6 was expressed in the AWA, AWB, ASH, and ADL neurons using cell-specific promoters introduced at multiple different concentrations. Intriguingly, the best restoration of str-2 expression was observed when GPA-6 was expressed in the ADL neurons, using either the gpa-11 or the sro-1 promoter (Figure 3C; Table S3). No strong restoration was observed when GPA-6 was expressed in the AWC cells (Figure 3C; Table S3). These results suggest that GPA-6 acts predominantly in the ADL neurons to regulate str-2 expression in the AWC neurons.
UNC-36 is broadly expressed in neuronal and probably also muscle tissue (SCHAFER et al. 1996). In the embryo, it is predicted to act in one of the two AWC neurons to repress str-2 expression in response to lateral cell contact (TROEMEL et al. 1999). Furthermore, the downstream genes unc-43, nsy-1, and sek-1 have been shown to act in AWC to regulate functional asymmetry (SAGASTI et al. 2001; TANAKA-HINO et al. 2002). We expressed UNC-36 in the AWC neurons using the gpa-13 promoter, which resulted in strong str-2 expression in >60% of the animals (Figure 3D; Table S4 at http://www.genetics.org/supplemental/). Although we cannot rule out that UNC-36 functions in the ASH or ADF neurons, it seems plausible that UNC-36 functions in AWC to promote str-2 expression. This implies that in one cell type, AWC, UNC-36 has a dual, paradoxical role and both suppresses and promotes str-2 expression.
ASI expression of str-2 is regulated by the same genes that regulate AWC expression but involves different neurons:
Thus far, we considered str-2 expression in the AWC neurons. Adult animals also express str-2 weakly in both ASI neurons and Dauer larvae express str-2 strongly in the ASI neurons. ASI expression in adults is regulated by Dauer pheromone and a TGFß-signaling pathway, which regulates entry in the Dauer stage (PECKOL et al. 2001; NOLAN et al. 2002). Mutations in the TGFß homolog daf-7, the SMAD transcriptional regulator daf-3 and the nuclear hormone receptor daf-12 affect the expression of str-2 and other receptors in the ASI neurons in all developmental stages (REN et al. 1996; SCHACKWITZ et al. 1996; PATTERSON et al. 1997; RIDDLE and ALBERT 1997). In young unc-36; odr-3; gpa-5 gpa-6 larvae, faint ASI expression was detectable, but this disappeared in adult animals (results not shown). To test if the G-protein/Ca2+/MAPK network regulates str-2 expression in the ASI neurons, we examined str-2 expression in Dauer larvae.
str-2 expression in unc-36; odr-3; gpa-5 gpa-6 Dauer larvae was strongly reduced in the ASI neurons and mostly "off" in the AWC cells (Figure 5A; results not shown). This suggests that the same network of G proteins and Ca2+/MAPK-signaling genes regulates str-2 expression in the ASI neurons of Dauer larvae. We determined in which neurons the G-subunits and UNC-36 function by examining Dauer larvae of the transgenic strains that gave the best rescue of str-2 expression in the AWC neurons (Figure 3). Unexpectedly, str-2 expression in ASI was partially restored when odr-3 was expressed in the AWB or in the ASH neurons of Dauer larvae (Figure 5B; Table S5 at http://www.genetics.org/supplemental/). Expression of odr-3 in the AWA, AWC, or ADF neurons did not restore str-2 expression in ASI (Table S5). Furthermore, str-2 expression was partially restored when gpa-6 was expressed in the AWB neurons, but was not or was only weakly restored when gpa-6 was expressed in the AWA, ASH, or ADL neurons (Figure 5C; Table S5). str-2 expression was also restored using an integrated gpa-5 transgene, but it was not strongly restored using cell-specific gpa-5 constructs (Figure 5D; Table S5). Only gpa-5 expression in the AWA neurons weakly restored str-2 expression. Finally, str-2 expression could be clearly restored by expressing unc-36 in the AWC, ASH, and ADF neurons (Figure 5E; Table S5). These results suggest that the same G-protein/Ca2+/MAPK network regulates str-2 expression in the ASI neurons as in the AWC neurons, but that the individual genes do not all function in the same cells. ASI expression seems to require ODR-3 in the AWB and ASH neurons, instead of AWC, and GPA-6 in the AWB neurons, instead of ADL. The site of action of GPA-5 and UNC-36 seems unaltered.
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-subunits in regulating str-2 expression. Loss of str-2 expression in unc-36; odr-3; gpa-5 gpa-6 animals is suppressed by additional mutations in gpa-2, gpa-3, and gpa-13 (Figure 2A). To test if these mutations also suppressed the loss of str-2 expression in odr-1 mutants, we generated gpa-2 gpa-3 gpa-13; odr-1 animals. In these mutants, str-2 expression was "off" (Table 2), suggesting that odr-1 is epistatic to all six G-subunits in regulating str-2 expression. In the AWA and ASH neurons, transient receptor potential V (TRPV) channels are thought to act downstream of G-protein sensory signaling (COLBERT et al. 1997; ROAYAIE et al. 1998). Three TRPV channels have been shown to regulate odr-10 receptor gene expression in AWA (TOBIN et al. 2002). Of these, the OSM-9 protein is also expressed in the AWC cells, where it is required for adaptation to certain odorants (COLBERT and BARGMANN 1995). Thus far, no involvement of OSM-9 in regulating receptor expression in AWC has been described. Therefore, we examined str-2 expression in osm-9 mutants. In osm-9 single and unc-36; osm-9 double mutants, no change in str-2 expression was observed (Table 2). However, mutations in unc-36, osm-9, and odr-3, gpa-5, or gpa-6 severely reduced str-2 expression, while mutations in all five genes restored expression to almost wild-type levels (Table 2). This shows that OSM-9 regulates str-2 expression in a complex manner and possibly functions in both stimulatory and inhibitory G-protein pathways to regulate str-2 expression.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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-subunits in a redundant fashion. Second, we find that a signaling network involving signals in several sensory neurons, mediated by GPA-2, GPA-5, GPA-6, ODR-3, and the UNC-36/UNC-43/NSY-1 pathway, promotes str-2 expression. GPA-3 and GPA-13 suppress str-2 expression in this network.
We propose a model in which signals from different neurons converge in the AWC neurons in adult animals to regulate str-2 expression (Figure 6A). Our results confirm previous cell-specific rescue experiments (SAGASTI et al. 2001; TANAKA-HINO et al. 2002; CHUANG and BARGMANN 2005), which suggest that the Ca2+/MAPK pathway functions in the AWC neurons. In addition, ODR-3 and probably GPA-2 function in the AWC neurons. AWC is the only amphid neuron in which GPA-2 is expressed, but we cannot exclude a function in an interneuron (ZWAAL et al. 1997). Furthermore, str-2 expression in the AWC neurons is stimulated by GPA-5 signaling in the AWA neurons, GPA-6 signaling in the ADL neurons, and possibly weakly by ODR-3 signaling in the ADF neurons. The noncell-autonomous function of these G-subunits is substantiated by several observations. First, GPA-5 and GPA-6 expression has never been observed in the AWC neurons (JANSEN et al. 1999; LANS et al. 2004). Second, using promoter::gfp fusions and immunofluorescence, we showed that the promoters used for cell-specific expression in the AWA, ADL, and ADF neurons do not drive expression in the AWC neurons. Third, two different, unrelated promoters were used to show that expression of GPA-6 in the ADL cells restores str-2 expression in the AWC cells. Finally, expression of GPA-6 in the AWC cells did not restore str-2 expression to similar levels as did its expression in the ADL cells.
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Neural signaling:
A major question that emerges is how signals from the AWA, AWB, ASH, ADL, and ADF neurons influence gene expression in the AWC and ASI neurons. One possibility is that these signals are transmitted via synaptic signaling. However, the AWC neurons are innervated only by the ADL, ASE, and ASI neurons (WHITE et al. 1986; DURBIN 1987). Thus, only the GPA-6 signal originating from the ADL neurons can be transmitted directly to the AWC neurons. The ASI neurons are not innervated by any of the other amphid neurons. Therefore, we propose that signaling via synapses does not play a major role, unless indirect signaling via interneurons is involved.
Paracrine or endocrine signaling might be a better explanation for the noncell-autonomous regulation of str-2 expression. In C. elegans, the sensory neurons control many processes, including Dauer development, longevity, body size, and social feeding, by secreting endocrine signals such as TGFß homologs, insulin-like peptides, and various neuropeptides (REN et al. 1996; SCHACKWITZ et al. 1996; DE BONO et al. 2002; FUJIWARA et al. 2002; LI et al. 2003; ROGERS et al. 2003). The genome of C. elegans contains at least five TGFß homologs, 38 insulin-like genes, 23 FMRFamide-related (FaRP), and 151 non-FaRP neuropeptides, many of which are expressed in the sensory neurons (REN et al. 1996; SCHACKWITZ et al. 1996; MORITA et al. 1999; NATHOO et al. 2001; PIERCE et al. 2001; LI et al. 2003; KIM and LI 2004). Importantly, str-2 expression itself is regulated by the TGFß homolog DAF-7, which is secreted by the ASI neurons (NOLAN et al. 2002).
Downstream effector molecules:
In this study, we identify three genes that regulate str-2 expression probably downstream of the G-protein and Ca2+/MAPK signaling network (Figure 6C). First, all G proteins, including GPA-3 and GPA-13, could signal via the TRPV channel OSM-9, as loss of osm-9 in different G-mutant backgrounds either reduced or restored str-2 expression. Second, double-mutant analysis suggests that odr-1 is epistatic to unc-36, unc-43, tax-4 (TROEMEL et al. 1999), and the G-subunit genes. Therefore, odr-1 acts downstream of, or in parallel to, the G-protein/Ca2+/MAPK network, although at present it is unclear how odr-1 regulates str-2 expression. Third, a daf-3 mutation suppressed the loss of str-2 expression in unc-36; odr-3; gpa-5 gpa-6 animals, suggesting that daf-3 acts downstream of the G-protein/Ca2+/MAPK network. DAF-3 is a coSMAD transcription factor that is negatively regulated by the TGFß pathway to suppress Dauer formation (PATTERSON et al. 1997). In addition, DAF-3 suppresses gene transcription in the pharynx, probably by directly binding to DNA (THATCHER et al. 1999). At this point, it is unclear where daf-3 functions in relation to odr-1.
str-2 expression does not require sensory signaling:
Previous studies have shown that, in the sensory neurons of C. elegans, genes required for detecting sensory cues also regulate axon morphology and receptor gene expression. For example, mutations in the cyclic nucleotide-gated channel subunits tax-2 and tax-4 and mutations in genes that disrupt cilia function cause ectopic axon outgrowth (PECKOL et al. 1999). Furthermore, the TRPV channels OSM-9 and OCR-2, which mediate olfaction in the AWA neurons, are necessary for odr-10 receptor gene expression (TOBIN et al. 2002). Therefore, axon maintenance and gene expression are thought to be regulated by sensory activity.
In this study, we show that the G-subunits involved in odorant detection also regulate str-2 receptor gene expression in the AWC and ASI neurons. The response to most odorants depends on signaling via ODR-3 and GPA-3, which is modulated by stimulatory signaling via GPA-13 and inhibitory signaling via GPA-5 and GPA-2 (ROAYAIE et al. 1998; LANS et al. 2004). No clear function for GPA-6 was found in olfaction. Regulation of str-2 expression, however, involves different mechanisms, suggesting that str-2 expression is not regulated by olfactory cues. Consistently, mutations in osm-6 and dyf-8, which disrupt sensory cilia formation and sensory perception, do not affect str-2 expression. These results suggest that sensory activity is not required for str-2 expression.
The Ca2+/MAPK pathway has dual, opposite functions in regulating str-2 expression:
Previous genetic analysis suggests that in the embryo the two AWC neurons develop into functionally different cells due to the activity of the Ca2+ channel UNC-2/UNC-36 and the CaMKII UNC-43, which activate the MAPKK kinase NSY-1 and the MAPK kinase SEK-1 via the TIR protein TIR-1 (TROEMEL et al. 1999; SAGASTI et al. 2001; WES and BARGMANN 2001; TANAKA-HINO et al. 2002; CHUANG and BARGMANN 2005). As a result, str-2 is expressed in only one of the two AWC neurons. Mutations in these genes cause str-2 expression in both AWC neurons, demonstrating that they repress str-2 expression. In this study, we show that mutations in unc-36, unc-43, and nsy-1, but not in sek-1 or tir-1, in combination with mutations in gpa-2, odr-3, gpa-5, and gpa-6 cause a severe loss of str-2 expression. Thus, part of the Ca2+/MAPK pathway also promotes str-2 expression together with G-protein signaling. We postulate that, in the embryo, UNC-36-, UNC-43-, and NSY-1-mediated signaling activates SEK-1 via TIR-1 to define AWC asymmetry, while later, once asymmetry is established, these proteins activate other downstream effectors to regulate and maintain the expression of specific genes, including str-2. Interestingly, in embryos, str-2 is expressed in 10 cells, whereas in adults only in AWC and ASI (TROEMEL et al. 1999), suggesting that in additional cells mechanisms exist that regulate str-2 expression.
Signaling networks:
Thus far, we have not identified other receptors regulated by the same G-protein/Ca2+/MAPK network. However, preliminary data suggest that expression of the diacetyl receptor odr-10 is regulated by G proteins via different mechanisms (H. LANS, G. JANSEN, A. KAHN, and C. BARGMANN, unpublished results). Additional receptors might be regulated by signaling networks involving different G proteins and other signaling genes. Therefore, the expression of multiple G-subunits in the sensory neurons of C. elegans could be a way to specifically fine tune its sensory responses. The G-protein/Ca2+/MAPK network identified in this study might be one of the mechanisms by which this is accomplished.
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