A bilaterally symmetric pair of C. elegans gustatory neurons, left ASE (ASEL) and right ASE (ASER) (refer to Figure 2 for the positions of sensory neurons described in this review), senses a number of chemicals in a left/right asymmetric manner and coexpresses multiple rGCs, GCY-6 (Guanylyl CYclase), -7, -14, -19, -20, and -29, and GCY-1, -3, -4, -5, -19, -22, and -29, respectively (23). GCY-14 of ASEL is essential in sensing environmental alkaline pH (26). Ectopic expression of GCY-14 in other sensory neurons, ASG, ASI, and ASER, makes these neurons sensitive to alkaline pH. GCY-14 functions as a homodimer, like mammalian rGCs (Figure 1B). Histidine-174 of the GCY-14 ECD is required for the detection of alkaline pH. Deprotonation of this histidine residue by alkaline pH may cause conformational changes in the domain that activates intracellular GC. Activation of GCY-14 then opens cGMP-gated cation channels consisting of TAX-4 (abnormal chemoTAXis) (α) and TAX-2 (β) subunits (43, 44), resulting in Ca²⁺ entry into ASEL. This Ca²⁺ entry also involves EGL-4 (EGg Laying defective), a cGMP-dependent protein kinase (PKG) (45–47), TAX-6, a calcineurin A ortholog (48), and phosphodiesterases (PDEs) (30). A neuronal calcium sensor (NCS-1) (49), which is a calcium-binding protein related to vertebrate GCAPs and 74% identical to human frequenin (50), is not required for Ca²⁺ entry, but enables its downstream signaling, since chemotaxis of ncs-1 mutants to alkaline pH is deficient (26).

GCY-14 is also required for sensing increases in Na⁺ or Li⁺ concentrations (34) and is a direct sensor for an increase in NaCl concentrations (26). In contrast to alkaline pH sensation, histidine-174 does not play a role in detecting increases in NaCl concentration, suggesting that other residues of the ECD are responsible for sensation. Disruption of the ASER-expressed rGC gene, gcy-22, results in broadly defective chemotaxis to nearly all salts sensed by ASER (27, 34, 40). gcy-22 is also required for animal responses to the amino acid, methionine, which is primarily sensed by ASER (34). In contrast, disruption of other gcy genes results in highly salt-specific chemosensory defects (34). gcy-1 mutant animals show markedly decreased responses to K⁺, which is sensed by ASER. gcy-4 shows chemotaxis defects on gradients of Br⁻ and I⁻. GCY-4 and GCY-22 may exist as homodimers and heterodimers, with each homodimer retaining residual function, since a gcy-4;gcy-22 double mutant shows a stronger defective phenotype in chemotactic response to Br⁻ and I⁻ (34). Although all mammalian rGCs exist in a homodimeric form, but not in a heterodimeric form, it has been shown theoretically and experimentally that C. elegans rGCs, ODR-1 (ODoRant response abnormal) and DAF-11 (abnormal DAuer Formation), function as an obligate heterodimer (4, 26). gcy-6 mutant animals show a dramatic decrease in their ability to respond to Mg⁺ gradients, sensed by ASEL (34). However, except for GCY-14, it remains to be shown whether these GCYs are direct sensor molecules by ectopic expression of these rGCs in unrelated neurons.

Caenorhabditis elegans exhibits defined behavioral responses to thermal gradients, which evoke two distinct behaviors. The first is cryophilic movement, or migration toward cooler temperatures than the growth temperature. This is the dominant behavior when the ambient temperature exceeds the growth temperature and is achieved by means of a biased random walk (52–54). The second behavior, isothermal tracking, is observed when animals reach thermal zones that are within 2°C of the growth temperature (52, 54–57). These behaviors involve at least three pairs of sensory neurons, AFD, AWC, and ASI (53, 56, 58, 59). AFD neurons, whose sensory dendrites terminate in a specialized ending that is composed of a primary cilium and an extensive array of microvilli (51, 60), depolarize and hyperpolarize upon warming and cooling, respectively, and temperatures warmer than the growth temperature drive cryophilic thermotactic behaviors (61). The thermosensory responses appear to be AFD cell-intrinsic properties, although surrounding glial cells tune thermal responses of AFD (62, 63).

At least eight genes are essential for thermotransduction by AFD: gcy-8, gcy-18, gcy-23, tax-4, tax-2, pde-2, and ncs-1. The three rGCs, GCY-8, GCY-18, and GCY-23, are exclusively expressed in AFD (37, 64), and participate in isothermal tracking (59, 65) and migration in linear thermal gradients (66). Recently, it has been shown by ectopic expression in diverse cell types that these rGCs are indeed direct thermosensor molecules, and that both the extracellular and ICDs are necessary for sensation (28). The TAX-4/TAX-2 cGMP-gated cation channel is also expressed in AFD and is essential for thermotaxis (37, 43, 44, 61). In AFD, pde-2 and pde-5, but not pde-1 or pde-3, are expressed, and pde-2, but not pde-5, mutations increase the threshold temperature for the activation of thermoreceptors and augment the thermoreceptor current lifetime (67). NCS-1 is also expressed in AFD and regulates behavioral responses to thermal gradients (49). As in pde-2 mutants, loss of NCS-1 prolongs thermoreceptor currents and elevates their threshold. Unlike loss of PDE-2, however, loss of NCS-1 increases voltage-activated outward currents (67). These results suggest that cGMP concentrations and NCS-1 help set the threshold temperature of rGCs in AFD.

Caenorhabditis elegans AMsh glial cells ensheathe neuronal receptive endings of 12 neurons, including AFD. It has been recently shown how glia control shapes of neuronal receptive endings through inhibition of an rGC expressed on the neuronal surface (68). KCC-3, a K⁺/Cl⁻ cotransporter, localizes specifically to the glial microdomain surrounding AFD receptive ending microvilli, where it regulates K⁺ and Cl⁻ levels. Cl⁻ ions act as a direct inhibitor of the GCY-8 rGC on the AFD receptive ending microvilli via Cl⁻ binding to the receptor’s ECD. GCY-8 has basal activity, which is inhibited by extracellular Cl⁻ ions released from AMsh glial cells. Without the inhibition, an increased level of cGMP at the neuronal receptive ending promotes the disappearance of AFD receptive ending shapes. Similarly, a pde-1 pde-5 double mutant displays a fully penetrant loss of AFD receptive endings. PDE-1 and PDE-5 are the only PDEs required for extension of AFD microvilli and appear to function redundantly. It appears that higher levels of cGMP at the AFD neuronal receptive ending antagonize WSP-1/Wiskott–Aldrich syndrome protein, an actin regulator that promotes microvillus formation through nucleation of actin filaments in the receptive ending (68). However, it remains to be clarified how cGMP antagonizes WSP-1 activity.

Carbon dioxide is detected by animals as an environmental cue that indicates the presence of food, predators, or mates, and as an internal cue that reflects internal metabolic state (69, 70). C. elegans has a pair of CO₂-sensing neurons, the BAG neurons, which mediate avoidance of CO₂ by adults and attraction to CO₂ by dauer larvae (71, 72). Adult C. elegans animals display an acute avoidance response upon exposure to CO₂ that is characterized by cessation of forward movement and rapid initiation of backward movement. This CO₂ avoidance is mediated by a cGMP signaling pathway that includes the cGMP-gated heteromeric channel TAX-4/TAX-2 (71). The GCY-9 rGC is responsible for CO₂ sensitivity of the BAG neurons (27). CO₂ avoidance behavior is modulated by multiple signaling molecules, including the neuropeptide Y receptor, NPR-1, and the calcineurin subunits, TAX-6 and CNB-1 (CalciNeurin B) (71). GCY-9 is a direct sensor for molecular CO₂, since the receptor ectopically expressed in AFD thermosensory neurons, which normally do not respond to CO₂, responded to CO₂ (27).

Caenorhabditis elegans uses two pairs of ciliated olfactory neurons, AWA and AWC, to sense many volatile attractants (39). A pair of laterally symmetric AWC neurons, AWCL and AWCR, are functionally distinct from each other. The distinction between the two AWC neurons is random from animal to animal, but coordinated so that one neuron of each type is generated in each animal (73). AWC^on, which expresses the STR-2 (Seven Transmembrane Receptor) G protein-coupled receptor (GPCR), detects butanone, benzaldehyde, and isoamyl alcohol, whereas AWC^off, which does not express STR-2, detects 2,3-pentanedion, benzaldehyde, and isoamyl alcohol (74). Chemosensation in AWC is mediated by direct binding of odorants to GPCRs, which are exclusively localized to sensory cilia (75, 76). Intracellularly, these receptors are coupled to heterotrimeric G proteins, whose α subunit can positively or negatively regulate C. elegans chemotaxis to odorants (77). There are at least four Gα subunits expressed in AWC: ODR-3, GPA-2 (G Protein, Alpha subunit), GPA-3, and GPA-13 (77–79). Butanone signaling is mediated by two partly redundant Gα proteins, ODR-3 and GPA-2, whereas other odor responses in AWC are mediated by ODR-3, but not by GPA-2 (78). AWC utilizes cGMP as a second messenger, as well as the cGMP-gated cation channel TAX-4/TAX-2. ODR-1 and DAF-11 rGCs are both required for the animal’s chemotaxis to all AWC-sensed odorants (31, 33, 39), and these rGCs form obligate heterodimers (Figure 1C), each monomer of which provides essential catalytic residues to form a single catalytic site (4, 26). The ECD of ODR-1 is dispensable for its olfactory function, indicating that the rGC does not act as a direct receptor for odorants sensed by AWC (31).

Genetically encoded calcium indicators have revealed that in the absence of odorants, the AWC exhibits high intracellular calcium levels, and upon odorant stimulation, intracellular calcium levels decrease, leading to hyperpolarization of the AWC neurons (80). This hyperpolarization can be explained by the decrease of cGMP levels followed by closing of TAX-4/TAX-2 channels. This mechanism shares many similarities with the light response in mammalian photoreceptor cells, where in the absence of light, calcium channels are open, exhibiting a “dark current.” Upon photon binding, a cGMP PDE is activated, which reduces cGMP levels, closing cGMP-gated channels, and thus hyperpolarizing the cell (81). Therefore, in AWC olfactory sensory processing in C. elegans, GPCRs act as sensor molecules, and upon stimulation, release their Gα subunits to regulate cGMP concentration in AWC cilia. Interestingly, C. elegans chemotaxis to odorants sensed by AWC is not affected by loss of the PDEs (82). Therefore, the released Gα may directly interact with the heterodimeric ODR-1/DAF-11 rGC to lower the GC activity (Figure 1C) (31).

The EGL-4 PKG is necessary for adaptation of the AWC chemosensory response (47, 83). Nuclear localization of EGL-4 is both necessary and sufficient to promote long-term adaptation (84) and is dependent on PDE activity (82). ODR-3, a Gα subunit, and the ability of EGL-4 to bind cGMP are both required for nuclear entry of EGL-4 after prolonged odorant exposure. Furthermore, loss of ODR-1 leads to constitutive entry of EGL-4 into the nucleus (83). Nuclear EGL-4 phosphorylates HPL-2 (Heterochromatin Protein Like), a heterochromatin-binding protein, and promotes the phosphorylated protein to bind to the odr-1 locus in AWC in order to reduce odr-1 mRNA levels in adapted animals. Concomitantly, the increased activity of an endo-siRNA pathway-targeted odr-1 degrades the mRNA (85).

An attractive behavior mediated by AWC^on requires the GCY-28 rGC, which acts in adults and localizes to the AWC^on axon. Mutations in gcy-28 lead to an avoidance behavior instead of the attractive behavior normally directed by the AWC^on neuron. This behavioral reversal results from presynaptic changes in AWC^on possibly through modification of AWC^on excitability or synaptic release by GCY-28 (41). The gcy-28 mutants also show an abnormal bias in the behavioral choice between two conflicting cues, the attractive odorant diacetyl sensed by AWA and the aversive stimulus Cu²⁺ sensed by ASH/ADL, although their responses to each individual cue are similar to those in wild-type animals (42). GCY-28 regulates the neuronal activity of AIA interneurons, where the conflicting sensory cues from AWA and ASH/ADL sensory neurons seem to converge, by activation of CNG-1, a cyclic nucleotide-gated ion channel (42, 86).

The environment determines whether C. elegans grows directly into adulthood, or whether it arrests at an alternative L3 larval stage to form a dauer larva (87). Dauer larvae are induced by harsh conditions, such as starvation and high population density, and can survive under severe conditions because of distinctive morphology, metabolism, and life span. When environmental conditions improve, dauer larvae reenter the reproductive cycle by molting into L4 larvae and subsequently into adulthood.

The dauer larva versus reproduction and growth decision is determined by at least two signaling cascades: the DAF-2 (insulin/IGF-1 receptor) (88) and the DAF-7 (TGFβ) pathways (89, 90). A decrease in either of the signals causes dauer arrest, indicating that both pathways are required for reproduction and growth. Downregulation of DAF-2 results in the activation and nuclear localization of DAF-16 (forkhead transcription factor) (91–93). The DAF-2 signaling pathway also regulates metabolism and aging. When DAF-2 signaling is decreased, life span is greatly extended (94, 95). daf-7 encodes a member of the TGFβ superfamily that is a ligand for the parallel neuroendocrine pathway (89, 90). DAF-7 activates a heterodimeric receptor consisting of DAF-1 (TGFβ type I receptor) (96, 97) and DAF-4 (TGFβ type II receptor) (98).

Laser ablation of three sensory neurons, ADF, ASG, and ASI, causes dauer arrest, indicating that these neurons signal reproduction and growth (99). Mutations in daf-11, which is expressed in ciliated sensory neurons including ASI, ASJ, ASK, AWB, and AWC, cause dauer arrest (33). Moreover, cGMP-gated channels have also been implicated (43, 44, 100). Because dauer arrest caused by a loss-of-function mutation, daf-11(lf), can be partially suppressed either by daf-3(lf) or daf-16(lf), DAF-11 is likely to regulate both the DAF-2 and DAF-7 pathways (101, 102). Furthermore, dauer-inducing pheromone inhibits DAF-7 expression and promotes dauer arrest, and food activates DAF-7 expression (89, 90). daf-7 gene expression is defective in daf-11 mutants, and the constitutive dauer formation phenotype of daf-11 mutants is suppressed by DAF-7 expression in ASI (103).

Two GPCRs, SRG-36 (Serpentine Receptor, class Gamma) and SRG-37, are strongly expressed in ASI neurons, where they localize to the sensory cilia, as receptors for the dauer pheromone, ascaroside ascr#5 (32, 104). A heterodimeric GPCR, DAF-37/DAF-38, is expressed in ASI and functions as a receptor for ascr#2 (105). odr-1 mutants have reduced sensitivity to dauer pheromone, indicating a role in ascaroside perception (32). While egl-4(lf) mutations increase the propensity to form dauer larvae (106), egl-4(gf) mutations decrease that propensity (107).

Taken together, at high population density, concentrations of dauer pheromone ascarosides increase and activate GPCRs on the cilia of ASI and other neurons. Gα released from the receptors may interact with heterodimeric ODR-1/DAF-11 rGC to regulate GC activity.

Caenorhabditis elegans lives in darkness, but is able to sense blue or shorter wavelength of light (with maximal responsiveness to ultraviolet light), and engages in negative phototactic behavior (108). This negative phototaxis is important for survival because ultraviolet and blue light are toxic to the animal on the soil surface and requires lite (light-unresponsive) genes (108). Laser ablation of a combination of four ciliated neurons (ASJ, AWB, ASK, and ASH) led to a severe defect in the negative phototaxis from light (109).

Caenorhabditis elegans phototransduction requires LITE-1, a seven-transmembrane domain receptor-like protein that transduces light signals in ASJ via G protein signaling (30, 110). C. elegans Gα proteins, GOA-1 and GPA-3, have a redundant role in mediating phototransduction in ASJ. Downstream of the Gα proteins, the rGCs ODR-1 and DAF-11 are required for phototransduction in ASJ. cGMP-gated cation channels consisting of TAX-4/TAX-2 act downstream of ODR-1/DAF-11. In the pde-1, -2, and -5 triple mutant, the photocurrent is markedly potentiated, with a current density about fivefold greater than that in wild-type animals. The photocurrent in pde-1, -2, -3, and -5 quadruple mutants exhibits very slow or no recovery after cessation of the light stimulus, consistent with a role for PDEs in downregulating cGMP levels. These results are reminiscent of vertebrate photoreceptor cells, where light-activated G proteins either inhibit PDEs (e.g., parietal eye photoreceptor cells) or stimulate them (e.g., rods and cones), increasing or decreasing cGMP levels and opening or closing cGMP-gated cation channels, respectively (30).

Many mutants that lack normal sensory cilia exhibit small body size, suggesting that sensory cilia are involved in regulation of body size in C. elegans (111). To elucidate molecular mechanisms underlying sensory regulation of body size, a genetic screen for suppressors of the small body size of a cilium-defective mutant, che-2 (abnormal CHEmotaxis), which is expressed in the cilia of most ciliated sensory neurons and which encodes a protein that contains G protein β-like WD-40 repeats has been carried out (112). Through the genetic screen, mutants defective in daf-25, egl-4, and gcy-12 have been isolated as suppressors of che-2 mutations. EGL-4 appears to regulate body size by functioning in sensory neurons, because EGL-4 expression in ASE (and AWC) sensory neurons is sufficient for the animal’s growth to a normal body size (38, 45). dbl-1 (DPP/BMP-Like) mutants have a small body size with low hypodermal ploidy. The DBL-1 (TGFβ) signaling pathway, which regulates hypodermal ploidy and consequently affects cell growth and body size (113), acts downstream of EGL-4 (45, 46). Indeed, che-2 and egl-4 mutants exhibit low and high ploidy of the hypodermal cells, respectively (114).

As described above, mutations in gcy-12 suppress the small body size of the che-2 mutant. As observed in egl-4 mutants, gcy-12 mutants showed increased body size, compared with wild-type animals (38). However, gcy-12;egl-4 double mutants did not become larger than egl-4 single mutants, indicating that gcy-12 and egl-4 act in the same pathway. The overexpression of GCY-12 in wild-type animals led to a small body size, presumably due to activation of EGL-4 kinase because GCY-12 overexpression did not cause body-size reduction in egl-4 mutants (38). These results also suggest that GCY-12 acts upstream of EGL-4, most likely by synthesizing cGMP. However, the ECD of GCY-12 is not necessary for GCY-12 function in body-size regulation (38). The pde-2 gene, which encodes a PDE is also required for normal body size, and pde-2 mutations cause a small body-size phenotype. The pde-2 mutation did not affect the body size of egl-4 mutants, indicating PDE-2 acts upstream of EGL-4. The gcy-12;pde-2 double mutants exhibited an intermediate body size relative to the two single mutants. These results indicate that PDE-2 hydrolyzes cGMP and negatively regulates EGL-4 kinase in regulating body size (38). A gain-of-function mutation of egl-4 that produces a constitutively activated EGL-4 kinase, exhibited small body size, an opposite phenotype of a loss-of-function mutant of egl-4 (115).

These results suggest the existence of other sensor molecules in addition to GCY-12 that detects extracellular signals. GCY-12 may be constitutively active in ASE. The extracellular signal may lower cGMP levels in ASE sensory neurons perhaps through activation of PDEs and consequently inhibits EGL-4 kinase. Abnormal activation of EGL-4 by an increased concentration of cGMP or by a mutation may upregulate the secretion of DBL-1 (also known as CET-1) or equivalent ligand molecules from ASE sensory neurons. An increase in DBL-1 concentrations may initiate the Sma/Mab signaling pathway in hypodermal cells, which regulates hypodermal ploidy and consequently affects cell growth and body size. In analogy to the role for EGL-4 in olfactory adaptation, in which loss of ODR-1 rGC leads to constitutive entry of EGL-4 into the nucleus of AWC, loss of GCY-12 rGC may also lead to entry of EGL-4 into the nucleus of ASE, where DBL-1 expression may be regulated.