Abstract
Sensory cues regulate several aspects of behavior and development in Caenorhabditis elegans, including entry into and exit from an alternative developmental stage called the dauer larva. Three parallel pathways, including a TGF-β-like pathway, regulate dauer formation. The mechanisms by which the activities of these pathways are regulated by sensory signals are largely unknown. The gene egl-4 was initially identified based on its egg-laying defects. We show here that egl-4 has many pleiotropies, including defects in chemosensory behavior, body size, synaptic transmission, and dauer formation. Our results are consistent with a role for egl-4 in relaying sensory cues to multiple behavioral and developmental circuits in C. elegans. By epistasis analysis, we also place egl-4 in the TGF-β-like branch and show that a SMAD gene functions downstream of egl-4 in multiple egl-4-regulated pathways, including chemosensation.
ORGANISMS make complex behavioral and developmental decisions on the basis of sensory cues in their environment. The nematode Caenorhabditis elegans responds to multiple types of sensory signals, including chemical, mechanical, and thermal stimuli (for recent reviews, see Driscoll and Kaplan 1997; Troemel 1999). Responses to these stimuli are mediated by well-defined circuits consisting of ciliated sensory neurons, interneurons, and motor neurons (Whiteet al. 1986). Functions of individual sensory neurons have been defined by laser killing experiments, and it has been shown that distinct subsets of sensory neurons are required for the response to each type of stimulus (Chalfieet al. 1985; Bargmann and Horvitz 1991a; Bargmannet al. 1993; Kaplan and Horvitz 1993; Mori and Ohshima 1995).
Several aspects of C. elegans behavior and development are regulated by environmental signals. Chemical cues direct movement toward sources of food and away from toxic compounds. Behaviors such as locomotion, pharyngeal pumping, defecation, foraging, and egg laying are also modulated by sensory cues (Horvitzet al. 1982; Avery and Horvitz 1990; Thomas 1990; Liu and Thomas 1994). In addition, environmental stimuli regulate developmental decisions such as entry into and exit from an alternative third larval stage called the dauer larva (for review, see Riddle and Albert 1997). Dauer larvae are specialized for survival and dispersal in harsh environmental conditions and can recover and resume reproductive growth when conditions improve.
The decision to enter into or recover from the dauer stage is made through the assessment of multiple parallel sensory and developmental inputs. A high concentration of a constitutively produced pheromone signals increased population density and is the primary chemosensory signal regulating dauer formation (Golden and Riddle 1982, 1984b, 1985). In addition, high temperature and low levels of food indicate adverse conditions and also promote dauer formation (Golden and Riddle 1984a,b). Thus, regulation of dauer entry and exit requires integration of information from multiple sensory pathways and provides an excellent model system in which to investigate several aspects of neuronal function.
Three signaling pathways that act in parallel to regulate dauer formation have been defined (Figure 1; Vowels and Thomas 1992; Thomaset al. 1993; Gottlieb and Ruvkun 1994; Riddle and Albert 1997). Most major players in each of these pathways have been identified by studying mutants that either enter the dauer state inappropriately under noninducing conditions [dauer-formation constitutive (Daf-c)], or fail to enter the dauer state under inducing conditions [dauer-formation defective (Daf-d)]. The group I Daf-c genes are thought to act by activating the ASJ chemosensory neurons among others to promote dauer formation under inducing conditions (Vowels and Thomas 1992; Thomaset al. 1993; Schackwitzet al. 1996). The group II Daf-c genes constitute a transforming growth factor-β (TGF-β)-like signaling pathway and function to repress dauer formation under noninducing conditions via the ASI, ADF, and ASG neurons (Bargmann and Horvitz 1991b; Schackwitzet al. 1996; Riddle and Albert 1997; Patterson and Padgett 2000). Under noninducing conditions, the DAF-7 TGF-β homolog is produced by the ASI neurons (Renet al. 1996; Schackwitzet al. 1996). The DAF-7 signal is transduced via the DAF-4 TGF-β type II and the DAF-1 TGF-β type I receptors, as well as the DAF-8 and DAF-14 SMAD proteins (Georgiet al. 1990; Estevezet al. 1993; Riddle and Albert 1997; Inoue and Thomas 2000). This pathway antagonizes the action of the DAF-3 SMAD protein and the daf-5 gene product (as yet uncloned) to repress dauer development (Pattersonet al. 1997). In a third pathway, an insulin-like ligand(s) represses dauer formation via the DAF-2 insulin receptor and the AGE-1 phosphatidylinositol-3-OH kinase (Morriset al. 1996; Kimuraet al. 1997). This pathway is antagonized by the DAF-18 PTEN phosphatase and the DAF-16 forkhead domain protein (Larsenet al. 1995; Linet al. 1997; Ogget al. 1997; Ogg and Ruvkun 1998; Gilet al. 1999; Rouaultet al. 1999). It is equally likely that these pathways act to promote reproductive growth as opposed to repressing dauer arrest. As shown in Figure 1, these pathways are thought to converge at the DAF-12 nuclear hormone receptor (Riddle and Albert 1997; Antebiet al. 1998). The signals from these parallel pathways are integrated via unknown mechanisms and transduced to result in coordinated developmental changes in multiple tissue types throughout the animal.
Parallel pathways mediate dauer formation. Genes described in this work are included in each pathway. GC, guanylyl cyclase; Hsp90, heat shock protein 90; TGF-βR, TGF-β receptor; NHR, nuclear hormone receptor; insulin R, insulin receptor; PI3 kinase, phosphoinositide-3-OH kinase. See text for additional details.
To reflect environmental changes accurately, it is crucial that the activities of the three major dauer regulatory pathways are appropriately regulated in response to sensory and developmental signals. The sensory cues pheromone, temperature, and food have been shown to regulate expression of the DAF-7 TGF-β ligand (Renet al. 1996; Schackwitzet al. 1996). Although the principal sensory neurons and genes regulating dauer formation have been identified, it is unclear how multiple sensory and developmental inputs are translated into modulation of each of these pathways. Since each branch of the dauer pathway is regulated by parallel sensory inputs, there is likely to be functional redundancy among genes that play roles in such regulatory events. One might also expect that these genes would function in several aspects of neuronal function and thus would show pleiotropic mutant phenotypes.
The genes unc-31, unc-64, and unc-3 were initially identified on the basis of the impaired movement of mutants (Brenner 1974). unc-31 and unc-64 mutants show pleiotropic phenotypes consistent with their general effects on neuronal function. unc-31 and unc-64 encode a CAPS-related protein and a syntaxin homolog, respectively; these proteins are required for calcium-regulated secretion (Livingstone 1991; Averyet al. 1993; Nguyenet al. 1995; Annet al. 1997; Ogawaet al. 1998; Saifeeet al. 1998). Mutations in these genes also affect the dauer pathway. While animals singly mutant in one of these genes form very few dauers under noninducing conditions, double mutant combinations with other genes result in a strong synthetic Daf-c phenotype (Syn-Daf; Ailionet al. 1999). The Daf-c phenotype of unc-31 and unc-64 mutants is strongly suppressed by mutations in daf-16, suggesting that these genes may regulate insulin release in response to sensory and/or metabolic signals (Ailionet al. 1999). Similarly, unc-3 mutants also show a number of pleiotropies, including a Syn-Daf phenotype (I. Katsura, personal communication; this work). unc-3 encodes an Olf-1/EBF1-like transcription factor that regulates expression of genes such as daf-7, as well as cholinergic genes in motor neurons (M. Ailion and J. H. Thomas, unpublished results; P. Ren and D. Riddle, personal communication; T. Starich and J. Shaw, personal communication; K. Lickteig and D. Miller, personal communication; Prasadet al. 1998). unc-3 functions in the TGF-β-mediated pathway for dauer formation, and the Daf-c phenotype of unc-3 mutants is fully suppressed by daf-3 and daf-5 mutations (M. Ailion and J. H. Thomas, unpublished results).
Studying genes with weak Daf-c phenotypes can therefore provide information about the mechanisms involved in the modulation of dauer regulatory signals, in addition to providing insight into specific aspects of neuronal function. Here we describe characterization of the gene egl-4. egl-4 was initially identified in screens for mutants defective in egg-laying behavior (Trentet al. 1983). We show that mutations in egl-4 result in several pleiotropies that include chemosensory defects, altered body length, and defects in synaptic transmission. We also find that egl-4 plays a role in relaying sensory cues to the egg-laying circuit. Moreover, we show that similar to unc-31, unc-64, and unc-3 mutants, egl-4 mutants are Syn-Daf and exhibit defects in dauer formation. Finally, we show that egl-4 mutations deregulate the TGF-β branch of the dauer pathway, and that most defects of egl-4 mutants, including chemosensory defects, are variably suppressed by mutations in daf-3 and daf-5. To our knowledge, this is the first report that implicates a SMAD protein in chemosensory signaling.
MATERIALS AND METHODS
Strains: Wild-type worms used were C. elegans variety Bristol, strain N2. Worms were grown using standard methods (Brenner 1974).
Strains carrying the following mutations were used in this work. Strains were obtained from the Caenorhabditis Genetics Center unless noted otherwise. Mutations are listed by linkage group:
LGI: daf-16(m27), daf-16(mgDf50), dpy-5(e61), egl-32(n155), unc-13(e450), unc-29(e1072), unc-75(e950).
LGII: daf-5(e1385), dpy-10(e128), kyIs37[odr-10-GFP::lin-15], tph-1(mg280), tra-2(q276), unc-4(e120), unc-52(e444).
LGIII: daf-2(e1370), daf-7(e1372), tax-4(ks11), unc-64(e246).
LGIV: daf-14(m77), egl-4(n477), egl-4(n479), egl-4(n612), egl-4(n579), egl-4(n478), flp-1(yn2), osm-3(p802), unc-31(e928).
LGV: daf-11(sa195), osm-6(p811).
LGX: daf-3(e1376), daf-12(m20), dpy-3(e127), kyIs53[odr-10-GFP(tagged)::lin-15], unc-1(e719), unc-3(e151), unc-6(e78), unc-58(e665).
flp-1(yn2) was obtained from C. Li; tph-1(mg280) was obtained from J. Y. Sze and G. Ruvkun; kyIs37 and kyIs53 were generated in the laboratory of C. I. Bargmann. The strain carrying mEx47-[daf-7-GFP::rol-6] was obtained from D. Riddle. unc-64(e246) was outcrossed once to remove an unlinked temperature-sensitive sterile mutation; the egl-4 alleles n477, n479, and n612 were outcrossed an additional two times before analysis. The following strains carrying multiple mutations were obtained from the Caenorhabditis Genetics Center: lin-1(e1275) unc-33(e204) IV, dpy-9(e12) ced-2(e1752) lin-1(e1275) IV. The following two strains were obtained from H. R. Horvitz: egl-32(n155) I; daf-3(e1376) X and egl-32(n155) I; daf-5(e1385) II.
Behavioral screens and assays: odr-9(ky27) and odr-9(ky185) were isolated in behavioral screens for mutants unable to chemotax towards diacetyl, essentially as described previously (Bargmannet al. 1993; Sengupta et al. 1994, 1996). Mutants were outcrossed a total of four times prior to further characterization. Population assays toward volatile and water-soluble chemicals were performed as described previously (Bargmann and Horvitz 1991a; Bargmannet al. 1993).
Noncomplementation between odr-9 and egl-4: odr-9(ky27) and odr-9(ky185) failed to complement for the defect in response toward diacetyl. odr-9(ky27) was mapped to the left arm of LGIV using standard mapping crosses. odr-9(ky27)/egl-4(n478) and odr-9(ky185)/egl-4(n478) trans-heterozygotes failed to complement for the phenotypes of chemotaxis defects, egg-laying defects, altered body size, darkened intestines, and hyperforaging behavior. All alleles of egl-4 were recessive for all phenotypes tested.
Egg-laying assays: To count and stage eggs, N2 and egl-4 adult hermaphrodites were mounted on agarose pads and viewed under Nomarski optics at ×400 magnification. Animals were grown at 20°, and first day adults were analyzed. To determine if egl-4 is responsive to food cues, single N2, egl-4(n478), and flp-1(yn2) adult hermaphrodites were placed on standard worm growth plates with either no food or a day-old lawn of bacteria. Animals were allowed to lay eggs for 2–3 hr at room temperature. Animals were then picked off the plate and the number of eggs laid was counted.
Dauer assays: Age-synchronized animals were allowed to lay eggs at room temperature for 3–6 hr. Parent animals were then removed and plates were incubated at the given assay temperatures. Dauer and nondauer animals were counted after ~100 hr at 15°, 65 hr at 20°, 48 hr at 25°, and 44 hr at 27°. This permitted the scoring of transient dauers that recover rapidly. Small differences in temperature >25° can make significant differences in the number of dauers formed, so each set of assays included all the relevant strains. All relevant comparisons are between strains assayed in parallel. Plates with partially purified dauer pheromone were prepared as described (Vowels and Thomas 1994). Additional details on the protocol followed for dauer assays are provided elsewhere (Ailion and Thomas 2000).
Serotonin assays: Serotonin was made as a 10 mg/ml stock solution in water and added to a final concentration of 1, 2, or 5 mg/ml to worm growth agar immediately before pouring. Plates were seeded with concentrated bacteria immediately before use. Dauer formation was assayed after 43 hr following synchronous egglays. After counting, plates were returned to 27° and incubated for an additional 4 days, after which the number of nondauers was counted to score for dauer recovery.
Aldicarb and levamisole assays: The effects of aldicarb and levamisole were scored in acute paralysis assays as follows. For both assays, plates were seeded with bacteria the day before the assay. A total of 20 young adult animals were picked to each of two duplicate plates. Aldicarb was made as a 100 mm stock solution in 70% ethanol and added to a final concentration of 0.5 or 1.0 mm to worm growth agar immediately before pouring. Animals were scored for movement and pharyngeal pumping when prodded with a platinum wire after 6, 8, and 10 hr. To most clearly show the differences between resistant and nonresistant strains, we plotted the percentage paralysis on 0.5 mm aldicarb at 10 hr, where paralyzed is defined as failure to move when prodded. Strains defined as resistant were clearly different from wild type at all time points and concentrations. Levamisole was made as a 100 mm stock solution in water and added to agar to a final concentration of 100 μm. Acute paralysis was scored every 30 min for 2 hr. Paralysis was defined as the absence of any moving or pumping when animals were prodded with a platinum wire.
Construction of double and triple mutant strains: Double mutants between egl-4 and various daf-c or daf-d mutations were constructed and confirmed by the methods described previously (Vowels and Thomas 1992; Thomaset al. 1993). Briefly, egl-4 double mutants with daf-c mutations were built by first constructing egl-4/+; daf-c/+ heterozygotes. egl-4 was homozygosed by picking Egl animals. Subsequently, the daf-c mutation was homozygosed by picking dauers and recovering them. egl-4 double mutants with daf-d mutations were built by constructing egl-4/+; daf-d/m heterozygotes where m is a visible marker. egl-4 was homozygosed by picking Egl animals, and the daf-d mutation was homozygosed by picking animals that failed to segregate the marker m. Markers used were as follows: daf-16—dpy-5(e61) unc-75(e950) or unc-13(e450); daf-3—unc-1(e719) dpy-3(e27); daf-5—unc-52(e444); daf-12—unc-58(e665) or unc-6(e78). The unexpected strong suppression of the 27° Daf-c phenotype of egl-4(n479) in several double mutants made us examine whether the 27° Daf-c mutation was actually present in the double mutants and hence whether the 27° Daf-c mutation was identical to the egl-4 mutation. First, we confirmed that the 27° Daf-c mutation in n479 mutants mapped to the same region as egl-4. Second, we deconstructed the egl-4(n479); daf-3 and daf-16; egl-4(n479) strains to reisolate the egl-4 mutation. In both cases, all egl-4 homozygotes generated were strongly Daf-c at 27°, indicating that the suppressed strains do contain the daf-c mutation and that it is tightly linked to egl-4.
An egl-4 osm-3 double mutant was constructed by first generating osm-3/egl-4 unc-33 heterozygotes. Egl non-Unc Osm recombinant progeny were selected and homozygosed. The egl-4; osm-6 double mutant was built by successively homozygosing egl-4 and osm-6 by the Egl and Osm or Dyf phenotypes, respectively. tph-1 doubles were built by picking Egl (egl-4) or Unc (unc-31, unc-64, or unc-3) animals segregating from tph-1/m; egl-4 or unc/+ heterozygotes, where m was dpy-10(e128) unc-4(e120), except in the case of the unc-3 double, where m was tra-2(q276). Animals that failed to segregate m were presumed to carry tph-1, which was also scored by a low-penetrance withered tail (Wit) phenotype. Triple mutants of egl-4; unc-3 with daf-5 or daf-16 were built by picking dauers from egl-4/+; unc-3/+; daf-5/unc-52 or daf-16/+ heterozygotes to homozygose both egl-4 and unc-3 simultaneously. After dauers recovered, daf-5 was homozygosed by picking animals that failed to segregate Unc animals, while daf-16 was homozygosed by picking partial dauers. The egl-32; egl-4 double mutant was constructed by crossing unc-13/+; egl-4/+ males with egl-32 hermaphrodites. Non-Egl cross-progeny were picked individually, and those segregating Unc animals were kept. Egl animals from these plates were again picked singly, and those segregating Unc animals were selected as animals having the genotype egl-32/unc-13; egl-4/egl-4. egl-32 was homozygosed by picking animals that failed to segregate Unc progeny. Presence of the appropriate single mutations was confirmed by complementation testing for visible or behavioral phenotypes. Additional details on strain constructions are available upon request.
The rationale behind the selection of alleles for some double mutant constructions is as follows. The tph-1(mg280), unc-3(e151), unc-31(e928), daf-11(sa195), daf-14(m77), and daf-16(mgDf50) alleles are likely null alleles (Averyet al. 1993; Ogget al. 1997; Prasadet al. 1998; Birnbyet al. 2000; Inoue and Thomas 2000; Szeet al. 2000). daf-7(e1372) mutants have been shown previously to exhibit a Daf-c phenotype equivalent in strength to that of animals carrying a predicted daf-7 null allele (Renet al. 1996). daf-2(e1370) results in severe loss of daf-2 function; no clear daf-2 null mutations have been identified (Kimuraet al. 1997). It has been shown previously that the Daf-c phenotype of daf-7(e1372) and daf-2(e1370) mutants is strongly enhanced in double mutant combinations with mutations in parallel branches of the dauer pathway, but not with mutations in the same branch (Thomaset al. 1993). daf-3(e1376) and daf-16(m27) alleles have been shown to suppress Daf-c phenotypes to a similar degree as the respective null alleles, and therefore likely represent strong loss-of-function mutations (Thomaset al. 1993; Gottlieb and Ruvkun 1994; Ogget al. 1997; Pattersonet al. 1997).
Statistical analysis: In all analyses involving comparisons among multiple groups, statistical significance was determined using the Bonferroni-Dunn multiple comparisons procedure, with the significance level set at 5%. Analyses were performed using the Statview 4.5 application (Abacus Concepts, Berkeley, CA).
RESULTS
odr-9 and egl-4 are allelic: We identified two alleles of the gene odr-9 (ky27 and ky185) in behavioral screens for mutants unable to respond to the volatile attractive chemical diacetyl (see materials and methods). We placed odr-9 in the same genetic interval as the previously identified gene egl-4 using standard three-factor mapping crosses (data not shown). Five alleles of egl-4 (n477, n478, n479, n579, and n612) have been identified in genetic screens for mutants with defects in egg-laying behavior (Trentet al. 1983). We found that odr-9 and egl-4 are allelic. n478/ky27 and n478/ky185 trans-heterozygotes fail to complement for all phenotypes tested (see materials and methods). This gene is henceforth referred to as egl-4. Here we present detailed characterization of seven alleles of egl-4.
egl-4 mutants are egg-laying defective: Since several alleles of egl-4 had been previously identified on the basis of their egg-laying defects, we further examined the egg-laying behavior of all egl-4 mutants. Egg-laying behavior has been described as biphasic, with periods of active egg laying interspersed with inactive periods (Waggoneret al. 1998). Induction of entry into the active phase is regulated by sensory cues and is mediated by the neurotransmitters serotonin and FMRFamide-related neuropeptides (Horvitzet al. 1982; Trentet al. 1983; Weinshenkeret al. 1995; Waggoneret al. 2000). Within the active phase, the rate of egg laying is regulated by an additional neurotransmitter, acetylcholine (ACh; Trentet al. 1983; Weinshenkeret al. 1995; Waggoneret al. 1998). It has been reported previously that egl-4 mutants exhibit normal rates of egg laying within the active phase, but have longer latent periods between active phases (Waggoneret al. 1998). Consistent with this, egl-4 mutants experience “transient bloating,” where animals become filled with eggs but eventually lay most of their eggs. In Table 1 we examined this egg-laying phenotype in two ways. First, we counted the number of eggs retained in the uterus of adult hermaphrodites and found that egl-4 mutants retain approximately three times as many eggs as wild-type adults of comparable stage. We also examined the developmental stages of eggs retained in the uterus of egl-4 mutants. Typically, early events in embryogenesis occur in utero; eggs are laid during gastrulation (at ~120–180 min postfertilization). We find that ~30% of the eggs retained in egl-4 mutants are at the comma stage or later in development (~400 min postfertilization or later). Eggs at this late developmental stage are rarely if ever observed in the uterus of well-fed wild-type hermaphrodites. Thus, egl-4 mutants lay eggs at a later developmental stage, likely as a consequence of delayed active egg-laying periods. All alleles appear to cause significant defects with no clear allelic series.
Number and stage of eggs in N2 and egl-4 animals
Since entry into the active phase of egg laying is regulated partly by serotonin, the defects of egl-4 mutants could result from pre- or postsynaptic defects in the serotonergic pathway. The egg-laying phenotype of egl-4 mutants is variably responsive to both serotonin and imipramine (a serotonin reuptake inhibitor; S. A. Daniels and P. Sengupta, data not shown; Trentet al. 1983), suggesting that egl-4 could function both pre- and postsynaptically. egl-4 could also act to potentiate the effect of serotonin on initiating the active phase of egg laying (Waggoneret al. 2000). Such a function has been ascribed to the FMRFamide-related neuropeptides encoded by the flp-1 gene. flp-1 plays a role in relaying sensory cues to the egg-laying circuit such that the rate of egg laying in flp-1 mutants is insensitive to food signals (Waggoneret al. 2000). To determine if egl-4 functions similarly, we compared the rate of egg laying in n478 mutants in the presence or absence of a bacterial food source. We find that egg laying by egl-4 mutants is insensitive to regulation by food cues (Figure 2). While egg laying by wild-type animals is significantly suppressed in the absence of food, the rate of egg laying in egl-4 mutants is unaffected. Thus, egl-4 may function to relay sensory cues to modulate the egg-laying circuit.
egl-4 mutants exhibit multiple defects in chemosensory behaviors: The ky27 and ky185 alleles were isolated on the basis of their failure to respond to the volatile odorant diacetyl. We examined the chemosensory behaviors of all egl-4 alleles, and show that egl-4 mutants have widespread chemosensory defects.
Attractive volatile chemicals: Attractive volatile chemicals are sensed by the bilaterally symmetrical ciliated neuron types AWA and AWC (Bargmannet al. 1993). AWA neurons mediate responses to the chemicals diacetyl (2,3-butanedione) and pyrazine, while AWC neurons are required for the attractive response to the volatile chemicals benzaldehyde, isoamyl alcohol, butanone, and 2,3-pentanedione. Both neuron types are required for the response to the chemical trimethylthiazole. Signaling molecules such as olfactory receptors, G proteins, and ion channels that function in each of these neuron types have been identified previously (Coburn and Bargmann 1996; Komatsuet al. 1996; Senguptaet al. 1996; Colbertet al. 1997; Zwaalet al. 1997; Roayaieet al. 1998; Jansenet al. 1999; Troemelet al. 1999).
Egg laying by egl-4 mutants is insensitive to food cues. Mean number of eggs laid per hour with or without food present is shown. The numbers of independent animals tested for each condition are indicated under the appropriate bars. Egg laying in wild-type animals is decreased in the absence of food (P < 0.001). Egg laying in egl-4 and flp-1 mutants is not significantly altered in the absence of food (P ≥ 0.05). P values were determined using the Mann-Whitney rank sum test.
We first examined the responses of all egl-4 mutants to odorants sensed by the AWA neurons. As shown in Figure 3, all egl-4 alleles tested exhibit very strong defects in the response to diacetyl and weaker but significant defects in the response to pyrazine. Since egl-4 mutants retain residual responses to pyrazine, egl-4 alleles likely affect a subset of functions rather than overall development of the AWA neurons. Diacetyl is recognized by the seven-transmembrane domain olfactory receptor ODR-10, which is expressed specifically in the AWA neurons and is localized to their sensory cilia (Senguptaet al. 1996). To determine if the strong diacetyl defect of egl-4 results from defects in odr-10 expression or localization, we examined expression and subcellular localization of a green fluorescent protein (GFP)-tagged ODR-10 fusion protein in n478 animals. Both expression and localization of ODR-10 were unaltered in n478 mutants (data not shown). We have shown previously that while odr-10 null mutants fail to respond to 1 nl of diacetyl, they respond relatively normally to 100 nl of diacetyl (Senguptaet al. 1996). Diacetyl (1 nl) is sensed exclusively by the AWA neurons, while higher concentrations are sensed redundantly by the AWA and AWC neurons (P. Sengupta and C. I. Bargmann, unpublished results). However, egl-4 mutants fail to respond to all concentrations of diacetyl tested (Figure 3D), consistent with defects in both the AWA and AWC chemosensory neurons.
Responses of egl-4 mutants to odorants sensed by the AWA neurons. Responses of egl-4 mutants to a point source of (A) 1 nl of diacetyl; (B) 1 μl of 10 mg/ml pyrazine; (C) 1 μl of 1:1000 dilution of trimethylthiazole; and (D) 100, 10, and 1 nl of diacetyl. Each data point represents the mean of at least six independent assays using ~200 animals in each assay. Error bars equal the SEM. Single asterisks mark responses that are different from wild type at P ≤ 0.01; double asterisks mark responses that are different at P ≤ 0.001. C.I., chemotaxis index.
We next tested the responses of egl-4 mutants to additional odorants sensed by the AWC neurons. While all alleles have normal responses to the odorant benzaldehyde, they have weaker defects in the responses to butanone and isoamyl alcohol, and strong defects in the response to 2,3-pentanedione, an odorant structurally related to diacetyl (Figure 4). Overall, n478 has the strongest defects and n477 has the weakest defects. All egl-4 mutants except n478 exhibit wild-type response to trimethylthiazole (Figure 3C).
Attractive water-soluble chemicals: C. elegans is also attracted to water-soluble chemicals such as NaCl and lysine (Ward 1973; Dusenbery 1974; Bargmann and Horvitz 1991a). This behavior is mediated largely by the ASE ciliated neuron type, with minor contributions from additional neurons (ASG, ASI, ADF, and ASK; Bargmann and Horvitz 1991a). In addition to wide-spread defects in responses to volatile attractive chemicals, we found that all egl-4 mutants have strong defects in their responses to NaCl and lysine (Figure 5). The morphology of a subset of ciliated neurons (ASI, ASK, ADL, AWB, ASH, and ASJ) can be visualized by filling animals with the lipophilic dye DiO (Perkinset al. 1986; Herman and Hedgecock 1990). Neurons of mutants with defects in the structure of the sensory cilia often fail to fill with dye (Perkinset al. 1986; Starichet al. 1995). However, egl-4 mutants dye-fill normally, and no obvious defects in the morphology of the neurons were visible (data not shown).
We also examined additional sensory behaviors. These included repulsion from volatile repellents, responses to mechanical cues such as nose touch and osmotic shock, and responses to gentle body touch. Sensory neurons mediating each of these behaviors have been identified (Chalfieet al. 1985; Bargmannet al. 1990; Kaplan and Horvitz 1993; Troemel et al. 1995, 1997). egl-4 mutants were found to be wild type in their responses to these stimuli (data not shown), indicating that mutations in egl-4 affect the functions of a restricted subset of neurons.
egl-4 mutants are hypersensitive to dauer-inducing conditions: Dauer formation is dependent on the perception of chemosensory cues such as dauer pheromone and food. These cues are sensed by ciliated neurons (Bargmann and Horvitz 1991b; Schackwitzet al. 1996). Mutants with chemosensory defects often have defects in the regulation of dauer formation (Lewis and Hodgkin 1977; Albertet al. 1981; Riddleet al. 1981; Thomas 1993; Vowels and Thomas 1994; Coburnet al. 1998). In addition to the chemosensory defects described above, egl-4 mutants also show defects in the dauer formation process. egl-4(n478) has been shown previously to be hypersensitive to dauer pheromone (Golden and Riddle 1984b). We verified this and extended it by demonstrating that all alleles of egl-4 exhibit hypersensitivity to dauer pheromone (Figure 6A). While wild-type animals make <1% dauers upon addition of 1 μl of dauer pheromone, at this concentration nearly 100% of egl-4(n479) animals form dauers. By this assay, the strengths of alleles are as follows: n479 > ky27, n579, n477 > n612 > n478, ky185. We note that this allelic order differs from that found for the response to volatile odorants.
Responses of egl-4 mutants to odorants sensed by the AWC neurons. Responses of egl-4 mutants to a point source of (A) 1 μl of 1:200 benzaldehyde, (B) 1 μl of 1:1000 dilution of butanone, (C) 1 μl of 1:100 dilution of isoamyl alcohol, and (D) 1 μl of 1:1000 dilution of 2,3-pentanedione. Each data point represents the mean of at least six independent assays using ~200 animals in each assay. Error bars equal the SEM. Single asterisks mark responses that are different from wild type at P ≤ 0.01; double asterisks mark responses that are different at P ≤ 0.001.
Responses of egl-4 mutants to water-soluble chemicals. Shown are responses of egl-4 mutants to 0.2 m NaCl and to 0.5 m lysine. Each data point represents the mean of five independent assays using ~100 animals in each assay. Error bars equal the SEM. All responses differ from wild type at P < 0.001.
Mutations in other Daf-c genes also result in hypersensitivity to dauer pheromone (Golden and Riddle 1984b; Thomaset al. 1993). However, unlike most of these mutants, which are strongly Daf-c at 25°, all egl-4 alleles except n479 form few or no dauers under noninducing conditions at this temperature. n479 exhibits a weak Daf-c phenotype at 25° (Figure 6A). Dauer formation is modulated by temperature, and it has been shown previously that several mutants with weak Daf-c phenotypes at 25° are strongly Daf-c at the elevated temperature of 27° (Ailionet al. 1999). Such mutants include unc-31, unc-64, and unc-3 (Ailionet al. 1999). We examined the Daf-c phenotypes of egl-4 mutants at 27°. As shown in Figure 6B, all egl-4 alleles exhibit a Daf-c phenotype to varying degrees at 27°. n479 is the strongest allele, forming close to 100% dauers at 27°, while ky185 is the weakest allele, forming <25% dauers. The allelic series with respect to dauer formation at 27° is roughly similar to that determined by pheromone hypersensitivity.
egl-4 is Syn-Daf with unc-3: Many weak Daf-c mutants have a Syn-Daf phenotype in double mutant combinations with unc-31, unc-64, and unc-3 mutants (I. Katsura, personal communication; Ailionet al. 1999). We wished to determine if egl-4 mutants are also Syn-Daf. egl-4 unc-31 and unc-64; egl-4 double mutants do not show Syn-Daf phenotypes (M. Ailion and J. H. Thomas, data not shown; I. Katsura, personal communication). However, we find that the egl-4(n478); unc-3(e151) double mutant is strongly Syn-Daf, forming nearly 100% dauers at all temperatures (see Table 5). unc-3 has been shown to regulate the expression of the DAF-7 TGF-β ligand (M. Ailion and J. H. Thomas, unpublished results; P. Ren and D. Riddle, personal communication). We examined the expression of a daf-7::GFP fusion gene in egl-4(n478) mutants, and found that unlike unc-3 mutants, mutations in egl-4 do not affect expression of the DAF-7 TGF-β ligand (data not shown).
Dauer formation phenotypes of egl-4 mutants. (A) The number of dauers formed at different concentrations of added pheromone is shown as a percentage of the total number of animals on the plate. Approximately 100–200 animals were counted at each concentration of pheromone. (B) Shown are the number of dauers formed after 2 days at 27°. See materials and methods for additional details. Approximately 100 animals of each genotype were counted. Numbers shown are from a single experiment. Experiments repeated on independent days show similar relative differences.
Serotonin acts in parallel to egl-4 to regulate dauer formation: egl-4 mutants exhibit a subset of the phenotypes associated with those of mutants with defects in serotonin signaling. For example, tph-1 tryptophan hydroxylase mutants that fail to synthesize serotonin have egg-laying defects and a weak Daf-c phenotype similar to that of egl-4 mutants (Szeet al. 2000). Serotonin has also been implicated in regulating foraging behavior and male mating (Loer and Kenyon 1993; Duerret al. 1999). We examined egl-4 mutants and found that they also have foraging and male mating defects (data not shown). This raised the possibility that egl-4 mutants have defects in serotonin signaling. The defects of tph-1 mutants can be rescued by the addition of exogenous serotonin (Szeet al. 2000). Although serotonin does not completely rescue the egg-laying defect of egl-4 mutants (see above), we tested whether exogenous serotonin could rescue the 27° Daf-c phenotype of the egl-4(n479) mutant. As shown in Figure 7A, serotonin does not rescue the 27° Daf-c phenotype of egl-4, unc-64, unc-31, or unc-3 mutants. However, serotonin does appear to enhance dauer recovery of these mutants. Exogenous serotonin leads to a dose-dependent increase in dauer recovery of egl-4, unc-64, and unc-3 mutants, and to a lesser extent of unc-31 mutants (Figure 7B). This suggests that the effect of serotonin on dauer recovery is not specific to egl-4.
To determine if serotonin acts in parallel to egl-4, we analyzed double mutants with tph-1 (Table 2). Dauer formation is enhanced in a tph-1; daf-7 double mutant, suggesting that tph-1 acts in parallel to the group II Daf-c pathway (Szeet al. 2000). Similarly, we find that the Daf-c phenotype of a tph-1; egl-4 double mutant is also strongly enhanced at all temperatures tested, indicating that tph-1 acts in parallel to egl-4 (Table 2). tph-1 mutants also enhance dauer formation of unc-31, unc-64, and unc-3 mutants, suggesting that tph-1 acts in parallel to these genes as well. We find that the tph-1; unc-31 and tph-1; unc-64 double mutants are no longer temperature sensitive for dauer formation, while the tph-1; egl-4 double mutant is weakly temperature sensitive.
egl-4 mutants exhibit synaptic transmission defects: unc-64 and unc-31 encode proteins that mediate synaptic transmission and other types of Ca2+-regulated secretion (Livingstone 1991; Averyet al. 1993; Annet al. 1997; Ogawaet al. 1998; Saifeeet al. 1998). Like egl-4, both mutants have multiple behavioral pleiotropies and are Syn-Daf. unc-64 and unc-31 are resistant to aldicarb, an inhibitor of acetylcholinesterase, indicating that these genes play a role in cholinergic transmission (Nguyenet al. 1995; Milleret al. 1996; Saifeeet al. 1998). To test whether egl-4 also plays a role in synaptic transmission, we examined the effects of acute exposure of egl-4 mutants to aldicarb. In Figure 8A, we show that the n479 and n612 mutants of egl-4 are strongly resistant to aldicarb while the ky27 mutant is less resistant. We also find that unc-3(e151) mutants are strongly resistant to aldicarb. In addition to the quantitative measurement of aldicarb resistance, egl-4 mutants are qualitatively less hypercontracted than wild-type animals on aldicarb.
egl-4 mutants and serotonin. (A) Shown is the percentage of dauers formed at 27° in the presence of 5 mg/ml serotonin in the plate. A total of 100–200 animals of each genotype were counted in two independent assays. (B) The number of dauers on plates containing serotonin were counted after 4 days at 27° to assay recovery. Wild-type animals recover in 1 day at 27° in the presence or absence of serotonin.
Resistance to aldicarb can arise either from a defect in presynaptic ACh synthesis or release, or from defects in the postsynaptic response to ACh. To determine whether egl-4 functions presynaptically or postsynaptically, we assayed sensitivity to the nicotinic ACh receptor agonist levamisole (Lewis et al. 1980a,b). A postsynaptic mutant unc-29(e1072) is completely resistant to levamisole (Fleminget al. 1997), whereas the unc-31(e928) mutant, which is involved in presynaptic release mechanisms, is sensitive. All three alleles of egl-4 tested are sensitive to levamisole, as are unc-3(e151) mutants, suggesting that egl-4 functions presynaptically in regulating synaptic transmission (Figure 8B). egl-4 mutants also appear to be hypersensitive to levamisole, similar to unc-31 mutants. However, unlike unc-31 mutants, egl-4 mutants exhibit adaptation at later time points, such that paralyzed animals resume pumping and slight movements of the nose.
egl-4 mutants have increased body length: Body length in C. elegans is regulated via a DPP/BMP-mediated signaling pathway (Savageet al. 1996; Padgettet al. 1998; Patterson and Padgett 2000). This pathway is similar to the TGF-β-mediated branch of the dauer pathway, but uses a different ligand and an independent set of receptors and SMAD signaling genes with the exception of the DAF-4 TGF-β type II receptor, which is shared by both pathways. daf-4 mutants are Daf-c and have reduced body length (Estevezet al. 1993). In contrast, we noted that egl-4 alleles cause increased body length (Figure 9). egl-4 mutants are ~20–30% longer than wild-type animals. n579 mutants show the strongest defect, being on average 31% longer than wild type, while the n477 and n612 mutants show the weakest phenotypes, being ~17% longer. In comparison, lon-2(e678) mutants, which have been implicated in the pathway regulating body length, are ~34% longer than wild-type animals.
egl-4 functions in the group II branch of the dauer signaling pathway: Three parallel pathways that regulate dauer formation have been identified (see Introduction). In addition to the Daf-c phenotype, egl-4 mutants share additional phenotypes in common with both group I and group II Daf-c genes. Group I Daf-c mutants exhibit chemosensory defects to volatile and water-soluble chemicals, while group II Daf-c genes have defects in egg laying, have dark intestines (Din), and exhibit a “clumpy” behavior, in which animals tend to congregate in clumps (Trentet al. 1983; Thomas 1993; Vowels and Thomas 1994; Riddle and Albert 1997). egl-4 mutants, in addition to having chemosensory and egg-laying defects, are also Din, but exhibit little if any clumpy behavior. To determine the pathway in which egl-4 functions, we made double mutants between egl-4 and mutations in each of these pathways. We examined suppression or enhancement of different phenotypes of egl-4 in these double mutants.
Dauer formation: The Daf-c phenotype of mutants that function in the group II pathway is fully suppressed by daf-3 and daf-5 mutations, while those in the other branches are not (Thomaset al. 1993). We first tested whether the dauer formation defects of egl-4 mutants are suppressed by daf-3 and daf-5. We found that mutations in either daf-3 or daf-5 completely suppress egl-4 dauer formation induced by pheromone (Table 3), suggesting that egl-4 functions in the group II pathway of dauer formation. We also assayed suppression of the Daf-c phenotype of the strong n479 allele at 27°. The Daf-c phenotype of n479 is suppressed by mutations in the Daf-d genes daf-3, daf-5, daf-16, and daf-12 (Table 4). daf-12 mutations suppress the dauer phenotypes of Daf-c mutants in all three branches, since these branches are thought to converge at daf-12 (Riddle and Albert 1997; Antebiet al. 1998). daf-16 mutations have been shown to suppress the 27° Daf-c phenotypes of mutants in different branches of the pathway (Ailionet al. 1999; Apfeld and Kenyon 1999). However, while dauer formation in unc-31 and unc-64 mutants is also suppressed by mutations in daf-12 and daf-16, the Daf-c phenotype of these mutants is not suppressed by daf-3 and daf-5 (Ailionet al. 1999). In contrast, as shown in Table 4, dauer formation in egl-4 mutants at 27° is strongly suppressed by daf-3 and daf-5 mutations, further confirming placement of egl-4 in the group II pathway. Interestingly, egl-4 and daf-3 appear to mutually suppress each other, since while the daf-3(e1376) mutant and egl-4(n479) each form dauers at 27°, the egl-4; daf-3 double mutant makes fewer dauers than either single mutant alone. Mutations in the Daf-d gene osm-6, which also lead to a Daf-c phenotype at 27° (M. Ailion and J. H. Thomas, unpublished results; Apfeld and Kenyon 1999), do not suppress egl-4, demonstrating that such mutual suppression is specific to daf-3.
tph-1 interactions with Syn-Daf genes
We also determined whether the Syn-Daf phenotype of the egl-4; unc-3 double mutant is suppressed by mutations in daf-5 and daf-16. We find that mutations in daf-5 completely suppress the egl-4(n478); unc-3(e151) synthetic dauer phenotype at either 15° or 25°, while daf-16 mutations fail to suppress at 25° and only weakly suppress at 15° (Table 5). These results further support placement of egl-4 in the group II branch.
Pharmacological analysis of synaptic transmission. (A) egl-4 mutants are resistant to aldicarb. The number of animals that are paralyzed on plates containing aldicarb is shown as a percentage of the total number of animals. Paralysis (defined as the absence of any movement or pumping when prodded) was scored after 10 hr on 0.5 mm aldicarb. Approximately 40 animals were counted for each genotype. (B) egl-4 mutants are sensitive to levamisole. Paralysis on 100 μm levamisole was scored every 30 min. Approximately 40 animals were counted for each genotype.
egl-4 mutants have increased body length. The mean body length (in millimeters) is shown for animals of the indicated genotypes. At least 30 adult animals of each genotype were measured under 100× magnification using Nomarski optics and an eyepiece micrometer. Animals were measured 24 hr after the final molt. The mean body length of each egl-4 mutant is different from that of wild type at P < 0.001.
Since dauer formation is regulated by parallel pathways, there is strong enhancement of the Daf-c phenotype in double mutants between genes in different pathways, but not between those acting in the same pathway (see Figure 1). For instance, while the Daf-c phenotypes of daf-8 and daf-11 mutants are incompletely penetrant at low temperatures, a daf-8; daf-11 double mutant forms 100% dauers (Thomaset al. 1993). In contrast, the phenotype of a daf-8; daf-14 double mutant is still incompletely penetrant since both of these genes function in the TGF-β pathway (Thomaset al. 1993). To further confirm that egl-4 functions in the group II pathway, we built double mutants of egl-4 with Daf-c mutations in different pathways and looked for enhancement of the Daf-c phenotype at 15°. At this temperature, egl-4 clearly enhances the Daf-c phenotypes of daf-11(sa195) and tax-4(ks11) (group I genes; Table 6). Although egl-4 does not enhance the Daf-c phenotype of daf-2(e1370) (a member of the third branch) at 15°, there is significant enhancement at 20°. These results suggest that egl-4 acts in parallel to the group I branch and the daf-2 branch. We find that dauer formation is also slightly increased in egl-4(n478); daf-7(e1372) and egl-4(n478); daf-14(m77) double mutants (Table 6). This is difficult to interpret since daf-7 and daf-14 are strongly Daf-c on their own at 15°. However, in both cases it appears that egl-4 does enhance the phenotype, suggesting that egl-4 may act at least partially in parallel to the TGF-β pathway, as well as the other two pathways. Since we do not know the molecular nature of the egl-4(n478) allele, it is formally possible that the enhancement observed in the double mutants is due to the nonnull nature of this allele.
daf-3 and daf-5 suppress egl-4 dauer formation on pheromone
Suppression of egl-4 dauer formation at 27°
Chemosensory behaviors: Group I Daf-c mutants exhibit numerous chemosensory defects (Vowels and Thomas 1994; Coburn and Bargmann 1996). Group I genes such as daf-11 encode components of a cGMP-mediated signaling pathway that function both in dauer formation as well as other chemosensory processes (Birnbyet al. 2000). To date, no genes in the TGF-β or the insulin pathway have been implicated in other chemosensory pathways (Trentet al. 1983; Renet al. 1996; Schackwitzet al. 1996; Szeet al. 2000; Tissenbaumet al. 2000). We further examined whether the chemosensory defects of egl-4(n478) are suppressed by daf-3 and daf-5 mutations. We find that daf-3(e1376) completely suppresses all olfactory defects of egl-4(n478) (Figure 10). However, the effects of daf-5 mutations are more specific. While daf-5(e1385) suppresses defects in behaviors mediated by the AWC olfactory neurons, it fails to suppress the diacetyl olfactory defect of egl-4, mediated by the AWA neurons. Interestingly, while neither daf-3 nor daf-5 mutants exhibit any olfactory defects of their own, daf-5(e1385) appears to enhance the relatively weak pyrazine response defect of n478. It is possible that this enhancement is specific for the e1385 allele of daf-5.
egl-4 and unc-3 synthetic dauer formation
To determine if mutations in other branches can also suppress the chemosensory behavioral phenotypes, we examined the sensory behaviors of double mutants between egl-4 and the Daf-d mutations daf-16(mgDf50) and osm-3(p802). Interestingly, we find that daf-16(mgDf50) exhibits chemosensory behavioral defects on its own (Figure 10). daf-16 mutants show severely reduced responses to the odorants butanone and isoamyl alcohol, and exhibit significantly reduced responses to pyrazine and trimethylthiazole. This is not specific to the daf-16 (mgDf50) allele, since daf-16(m27) mutants show similar defects (data not shown). Unlike daf-3 and daf-5, daf-16 mutations fail to significantly suppress any chemosensory defects of egl-4(n478) (Figure 10). However, like egl-4(n478); daf-5(e1385) double mutants, the egl-4(n478); daf-16(mgDf50) double mutant shows an enhancement of the defect in the response to pyrazine. The osm-3 mutation also largely fails to suppress the chemosensory defects of egl-4 mutants, showing only weak partial suppression for the response to butanone. Finally, we also examined the responses of egl-4(n478); daf-12(m20) double mutants. Although daf-12 mutations suppress the Daf-c phenotypes of mutants in all three branches of the dauer pathway, it has been shown previously that daf-12 fails to suppress other pleiotropies (Thomas 1993; Riddle and Albert 1997; Antebiet al. 1998), suggesting that daf-12 is the point of convergence only for the dauer pathway. Consistent with this, we find that daf-12 fails to suppress any of the behavioral defects of egl-4 mutants. Surprisingly, we also find that daf-12(m20) single mutants show strong defects in the response to butanone (Figure 10). These results provide additional evidence to indicate that egl-4 functions in the group II pathway, upstream of daf-3 and daf-5.
egl-4 interactions with Daf-c mutations
Egg-laying behavior: It has been shown previously that in addition to suppressing the Daf-c phenotype, daf-3 and daf-5 mutants suppress other pleiotropies of group II Daf-c genes. These include the egg-laying defect, the Din phenotype, and the clumpy behavior (Trentet al. 1983; Vowels and Thomas 1994). We reexamined whether the egg-laying defect of egl-4 mutants is suppressed by daf-3 and daf-5 mutations, and also determined whether this defect can be suppressed by mutations in other branches of the pathway. As shown in Figure 11A, we find that both daf-3(e1376) and daf-5(e1385) partially suppress the egg-laying defect of egl-4(n478) mutants. In contrast, osm-3, daf-16, and daf-12 double mutants clearly show less suppression. Qualitative examination also indicates that the Din phenotype of egl-4 is suppressed by daf-3 and daf-5, but not by daf-16, daf-12, or osm-3 (data not shown).
Body length: Mutations in the DAF-4 TGF-β receptor cause small body length, yet this defect is not suppressed by daf-3 and daf-5 mutations (Vowels and Thomas 1992). An independent set of SMAD genes functions downstream of DAF-4 to mediate body size regulation (Savageet al. 1996; Padgettet al. 1998). Since daf-3 and daf-5 suppress all phenotypes of egl-4 tested, we wished to determine if the increased body length phenotype could also be suppressed by daf-3 and daf-5 mutations. We found that daf-3(e1376) fully suppresses the increased body length of egl-4(n478) (P > 0.03 compared to wild type), whereas this phenotype is only partially suppressed by daf-5(e1385) (Figure 11B). We find that daf-16(mgDf50) and osm-3(p802) also show weak suppression. As reported previously, daf-12(m20) single mutants show dramatically increased body length (Thomaset al. 1993), and we find that daf-12 mutants are on average ~21% longer than wild-type animals. The effects of mutations in egl-4 and daf-12 are partially additive for body length since the egl-4; daf-12 double mutant is significantly longer than either single mutant (P < 0.001; Figure 11B). Taken together, these results are strongly consistent with the placement of egl-4 in the group II pathway, either together with the TGF-β Daf-c genes or partially in parallel.
Chemosensory phenotypes of egl-4 mutants are suppressed by daf-3 and daf-5 mutations. Chemotaxis indices between 0 and 1.0 have been divided into 11 equal-sized bins and assigned a circle of size indicated at the bottom. Open circles represent a negative chemotaxis index; filled circles represent a positive chemotaxis index. Each data point represents the mean of at least three independent assays of ~200 animals each. Concentrations of odorants used are as indicated in Figures 3 and 4. The alleles indicated for each single mutant are present in the double mutant strains. Responses significantly different from that of wild type at P < 0.01 are indicated by an asterisk next to the circle.
egl-32 may function in the same pathway as egl-4: A single egl-32 allele (n155) was isolated in the same screens that resulted in the isolation of egl-4 alleles (Trentet al. 1983). egl-4 and egl-32 appear to function at least partly in similar pathways since it has been shown that (1) both egl-4 and egl-32 show similar variable responses to exogenous serotonin and imipramine (Trentet al. 1983), (2) the egg-laying defects of both egl-32 and egl-4 mutants are suppressed by daf-3 and daf-5 mutations (Trentet al. 1983), and (3) egl-4 and egl-32 are Syn-Daf with unc-3 mutations (I. Katsura, personal communication). However, unlike egl-4, egl-32 is not hypersensitive to dauer pheromone and we find that egl-32 is not Daf-c at 27° (M. Ailion and J. H. Thomas, unpublished results; Golden and Riddle 1984b). In addition, egl-32(n155) does not enhance the Daf-c phenotype of egl-4(n478) (data not shown).
daf-3 and daf-5 suppress the altered body length and egg-laying phenotypes of egl-4. (A) The number of eggs in the uterus of animals of the indicated genotypes was counted. n ≥ 20 for each genotype. Asterisks denote differences from wild type at P < 0.01. (B) The mean lengths in millimeters of adult hermaphrodites are shown. At least 20 animals of each genotype were measured. Mean body lengths of single and double mutants that are significantly different (P < 0.001) from that of wild-type animals are indicated by an asterisk.
egl-32 mutants show chemosensory defects that are suppressed by daf-3 and daf-5. The representation of the data is as described for Figure 10. Each data point represents the mean of at least three independent assays of ~200 animals each. Responses significantly different from that of wild type at P < 0.01 are indicated by an asterisk next to the circle.
Since egl-4 mutants exhibit strong chemosensory defects, we examined the chemosensory behaviors of egl-32(n155) mutants. We find that egl-32 mutants show strong chemosensory defects in the responses mediated by the AWC neurons, with weak defects in the responses to odorants sensed by the AWA neurons (Figure 12). egl-32 mutants also exhibit weak defects in the responses to NaCl and lysine (data not shown). The chemosensory defects of egl-32; egl-4 double mutants do not appear to be significantly stronger than either mutant alone with the exception of partial enhancement of defects in the responses to 2,3-pentanedione and trimethylthiazole. This suggests that egl-32 functions partially in parallel to egl-4 with respect to some chemosensory behaviors. We also find that daf-3 and daf-5 suppress the olfactory defects of egl-32 mutants with the daf-5(e1385) mutation suppressing more strongly than the daf-3(e1376) mutation (Figure 12). Although these data should be interpreted with caution since only one mutant allele of egl-32 is available, the phenotypic analyses suggest that egl-4 and egl-32 may function partly similarly to regulate dauer formation, and chemosensory and egg-laying behaviors.
DISCUSSION
egl-4 mutants show a number of behavioral and developmental abnormalities, including defective chemosensation and dauer formation, defective egg laying, altered body size, and defective synaptic transmission. Most phenotypes of egl-4 mutants are suppressed by mutations in the daf-5 and daf-3 (encoding a SMAD protein) genes, suggesting that egl-4 functions through the group II branch of the dauer formation pathway to regulate multiple functions. We propose that egl-4 acts to relay sensory cues to modulate different neuronal signaling pathways.
egl-4 mutant phenotypes arise from defects in neuronal function: We have shown that mutations in egl-4 result in multiple behavioral and developmental abnormalities. These include defects in egg-laying behavior, chemosensory perception, dauer formation, and regulation of body size. These defects could, in principle, arise from functions of egl-4 in multiple tissue types. However, taken together, analyses of egl-4 phenotypes presented here indicate that the focus of egl-4 action is primarily neuronal. We provide several lines of evidence in support of this hypothesis.
All behavioral and developmental pathways affected by egl-4 mutations are regulated by neuronal inputs. Environmental cues are sensed by sensory neurons. Signals are then relayed to interneurons where integration of these signals occurs (for example, see Mori and Ohshima 1995). Interneurons then relay this information to target tissues. The relatively specific chemosensory defects of egl-4 mutants point to a deficit in sensory signaling, or perhaps integration, rather than a general defect in target tissues. This is also consistent with the effects of egl-4 mutations on dauer formation. The target of the DAF-7 TGF-β ligand in dauer formation is thought to be neuronal (Inoue and Thomas 2000). The coordinated developmental changes that occur throughout the animal in response to dauer cues indicate that integration of dauer-forming signals occurs in neurons, and a coordinated and integrated signal is then sent to additional tissue types. Mosaic analysis has shown that the DAF-4 TGF-β type II receptor functions nonautonomously in the nervous system for dauer formation (Inoue and Thomas 2000). Work presented here suggests that egl-4 functions with or partially in parallel to the group II Daf-c genes (see below), suggesting that egl-4 may also function in neurons.
Egg-laying behavior is also regulated by neuronal inputs. Serotonin is released primarily by the HSN neurons and allows egg-laying muscles to enter into an active state (Horvitzet al. 1982; Trentet al. 1983; Desaiet al. 1988; Weinshenkeret al. 1995; Waggoneret al. 1998). Within the active state, ACh released by both the HSN and VC motor neurons promotes egg laying by excitation of egg-laying muscles (Waggoneret al. 1998). The action of serotonin is potentiated by flp-1-encoded neuropeptides, which may act to relay sensory cues to the egg-laying machinery (Waggoneret al. 2000). Analysis of the egg-laying pattern of egl-4 mutants indicates that egl-4 is deficient in the induction of the active egglaying phase, similar to serotonergic mutants as well as flp-1 mutants (Waggoner et al. 1998, 2000). Similar to flp-1 mutants, egl-4 mutants are also unable to regulate their rate of egg laying in response to food cues. flp-1 is expressed in interneurons that integrate sensory information (Schinkmann and Li 1992). The defects of egl-4 are consistent with a role for egl-4 in sensory neurons and/or interneurons to integrate or relay sensory signals.
Body size may also be regulated by signals from neurons. The DBL-1 DPP/BMP-like signal that regulates body size is expressed in neurons (Suzukiet al. 1999). This signal is then transduced to other tissue types such as the hypodermis to regulate body size. egl-4 could function in neurons to modulate body size regulatory signals.
Finally, we have shown that egl-4 mutants are resistant to the acetylcholinesterase inhibitor aldicarb. This is a characteristic of genes that function at cholinergic synapses (Milleret al. 1996). Mutations in presynaptic genes such as unc-17, which encodes the vesicular ACh transporter, and cha-1, which encodes the choline acetyltransferase, are also aldicarb resistant due to reduced or absent ACh at the neuromuscular junction (Alfonso et al. 1993, 1994). unc-29 mutants are also aldicarb resistant since unc-29 encodes the α subunit of the nicotinic ACh receptor, which functions postsynaptically in target muscles (Fleminget al. 1997). However, we show that unlike unc-29 mutants, egl-4 mutants are levamisole sensitive, similar to unc-17 and cha-1. This strongly indicates that the focus of egl-4 function is neuronal. The relatively restricted phenotypes of egl-4 mutants suggest that egl-4 is required for function rather than development of the nervous system.
egl-4 and neurotransmitters: Our results indicate that egl-4 functions presynaptically, at least in cholinergic neurons. Both ACh and serotonin have been implicated in regulating dauer formation and recovery (Szeet al. 2000; Tissenbaumet al. 2000). This raises the possibility that egl-4 plays a role in the release or synthesis of neurotransmitters such as ACh or serotonin. In general, mutants with strong defects in ACh synthesis or release at the neuromuscular junction show movement abnormalities and are uncoordinated. These include unc-17, cha-1, unc-31, unc-3, and unc-64. unc-3 encodes a transcription factor that is required for the expression of unc-17 and cha-1 in cholinergic motor neurons (K. Lickteig and D. Miller, personal communication). unc-31 and unc-64 may be required for exocytosis of ACh. However, egl-4 mutants show no gross movement defects. It is possible that egl-4 affects ACh synthesis or release but to a lesser degree such that in egl-4 mutants the level of ACh released is sufficient to excite muscles, but insufficient to mediate the paralyzing effects of aldicarb. ACh regulates the frequency of egg laying in the active phase, and cha-1 mutations result in a decrease in the frequency of egg laying within the active phase (Waggoneret al. 1998). The frequency of egg laying in the active phase is unaffected in egl-4 mutants, consistent with a minor effect of egl-4 mutations on ACh release.
egl-4 mutants share a subset of defects found in mutants defective in serotonin signaling. These include defects in egg laying, foraging, male mating, and effects on dauer formation. However, our results suggest that egl-4 is not directly involved in the serotonergic pathway. First, added serotonin fails to fully rescue the egg-laying defects or dauer formation defects of egl-4 mutants. Rescue by serotonin would be expected if egl-4 functions presynaptically in serotonin signaling. Second, we find that the Daf-c phenotype of egl-4; tph-1 mutants is strongly enhanced, suggesting that tph-1 functions in parallel to egl-4 and not in the same pathway. Third, serotonin has been shown to inhibit ACh release at the neuromuscular junction (Lackneret al. 1999; Milleret al. 1999; Nurrishet al. 1999). Mutants with defects in the serotonin pathway exhibit hyperactivity and are aldicarb hypersensitive due to excessive release of ACh. egl-4 mutants, on the other hand, show no movement defects and are aldicarb resistant. These results suggest that egl-4 does not directly function in the serotonin pathway. However, it remains formally possible that egl-4 has weak effects in both the serotonergic pathway and the cholinergic pathway.
egl-4 and the group II Daf-c genes: egl-4 shares a number of phenotypic similarities with group II Daf-c genes. In addition to effects on dauer formation, these include defects in egg laying and a darkened intestine. However, the phenotypes of group II and egl-4 mutants are not identical. Group II mutants (with the exception of daf-4 mutants) also exhibit a clumpy behavior, a trait not exhibited by egl-4 mutants. In addition, egl-4 mutants have increased body size whereas among the group II genes, only daf-4 has altered body size. Unlike egl-4 mutants, daf-4 mutants are smaller than wild type. Also, while the egg-laying phenotype of egl-4 is variably sensitive to serotonin and imipramine, egg laying by the group II Daf-c mutants is sensitive to both of these agents (Trentet al. 1983). egl-4 mutants also share some behavioral defects in common with group I genes in that egl-4 mutants exhibit strong defects in chemosensory behaviors. No group II Daf-c genes show significant defects in chemosensation (S. Daniels and P. Sengupta, unpublished results; Vowels and Thomas 1994), although this behavior is difficult to measure due to their clumpy phenotype. It should be noted that the PDK-1 Akt/PKB kinase that functions in the insulin pathway also shares some mutant phenotypes with egl-4 and group II genes, including increased body size, egg-laying defects, and clumpy behavior (Paradiset al. 1999). These phenotypes are suppressed by daf-16 mutations, but not by daf-5, osm-6, or daf-12 mutations (Paradiset al. 1999).
The strongest evidence that egl-4 functions in the group II branch of the pathway is the fact that many phenotypes of egl-4, including chemosensory, dauer formation, egg laying, and body size defects, are suppressed by mutations in daf-3 and daf-5, but not by group I Daf-d or by daf-16 mutations. This is a characteristic of group II genes, since Daf-c genes in group I or the insulin pathways are not fully suppressed by daf-3 and daf-5. However, daf-3 and daf-5 mutations fail to suppress the aldicarb resistance phenotype of egl-4 mutants (data not shown).
The TGF-β branch appears to act partly in parallel to serotonin, since dauer arrest of daf-7 mutants is strongly enhanced by tph-1 mutations (Szeet al. 2000). We have shown that egl-4 and serotonin also act in parallel, since the dauer formation phenotype of egl-4 mutants is also enhanced by tph-1 mutations. This is consistent with a role for egl-4 in the group II branch. It is also possible that egl-4 functions partly in parallel to the TGF-β pathway with outputs to daf-3 and daf-5. This possibility could explain the fact that there appears to be some enhancement of the Daf-c phenotype of egl-4(n478) in double mutant combinations with group II Daf-c mutants such as daf-7 and daf-14.
The Daf-d genes daf-3, daf-5, daf-16, and daf-12 function in chemosensation: In addition to affecting dauer formation, genes involved in the TGF-β branch also regulate other aspects of nondauer development as is evident from pleiotropies in the mutants. Since all pleiotropies are suppressed by daf-3 and daf-5 mutations, daf-3 and daf-5 may function in multiple tissue types to antagonize the TGF-β pathway. The DAF-3 SMAD protein is expressed in multiple tissue types including neurons, pharynx, hypodermis, and intestine; these tissues are extensively remodeled during the dauer stage (Pattersonet al. 1997). It has been shown that DAF-3 represses gene expression in the pharynx by directly binding upstream of gene sequences (Thatcheret al. 1999). Remarkably, we find that daf-3 and daf-5 mutations also suppress chemosensory defects of egl-4 mutants. This suggests that daf-3 and daf-5 function downstream of egl-4 to integrate or modulate chemosensory information. However, since the egl-4 allele used in these experiments could be nonnull, we are unable to rule out the possibility that daf-3 and daf-5 act upstream of egl-4. In either case, this is an unexpected finding since neither daf-3 and daf-5 nor the group II Daf-c genes exhibit chemosensory defects on their own (this work; Vowels and Thomas 1994). We show that there appear to be some differences in the degree of suppression of egl-4(n478) by daf-3(e1376) and daf-5(e1385) with respect to different phenotypes. For example, daf-5(e1385) enhances the pyrazine defects of egl-4(n478), suggesting that daf-5 may function independently of the TGF-β pathway in the response to pyrazine. Also, while daf-3 suppresses all chemosensory defects of egl-4, suppression by daf-5 is restricted to responses mediated by the AWC neurons. Interestingly, daf-5 appears to suppress egl-32(n155) defects more strongly, suggesting that the DAF-3 SMAD gene and DAF-5 may have unique functions as well as distinct sites of action in regulating chemosensory responses.
Daf genes in other branches affect aspects of C. elegans development other than dauer formation. For example, the insulin pathway regulates life span such that daf-2 mutants have extended life span (Kenyonet al. 1993). This effect is suppressed by loss-of-function mutations in daf-16 (Kenyonet al. 1993). We show that daf-16 and daf-12 Daf-d mutants also have chemosensory defects. This may occur due to deficits in downstream processing events, or possibly from defective regulation of genes directly involved in sensory perception. These results implicate these Daf-d genes in additional signaling pathways.
Model for egl-4 function: The effect of egl-4 on different pathways clearly arises from deregulation of neuronal functions, several of which are regulated by sensory cues. These include chemosensory behaviors, dauer formation, foraging, and egg laying. Signals from food sources are attractive and they promote nondauer development, increase egg laying, and inhibit foraging. As initially proposed by Trent et al. (1983), it is possible that egl-4 is involved in relaying sensory cues to these pathways, thereby modulating these circuits.
Sensory cues are known to regulate each of the dauer formation pathways. daf-7 is expressed in the ASI neurons and is downregulated in response to high pheromone, low food, and high temperatures (Renet al. 1996; Schackwitzet al. 1996). It is possible that these sensory cues are directly sensed by ASI to regulate daf-7 expression. However, it is also likely that cues sensed by other sensory neurons, in addition to ASI, are transmitted to regulate daf-7 expression (Kogaet al. 1999; Szeet al. 2000). Since we have not observed a direct effect on daf-7 expression in egl-4 mutants, egl-4 could act downstream of the daf-7 expression step by either modulating the action of DAF-7 and other TGF-β pathway genes in response to environmental signals or by modulating the activity of the daf-3 and daf-5 genes.
Similarities between egl-4 and the Daf-c genes in the TGF-β pathway suggest that these genes may act together or in parallel to relay sensory cues to the egg-laying circuit. Mutants in both egl-4 and the Daf-c genes in the TGF-β pathway display egg-laying defects and exhibit long intercluster intervals (L. Hardaker and W. Schafer, personal communication; Trentet al. 1983; Waggoneret al. 1998). In addition, similar to egl-4 mutants, egg laying by TGF-β pathway Daf-c mutants is insensitive to food signals (L. Hardaker and W. Schafer, personal communication). Finally, both daf-3 and daf-5 mutations suppress the egg-laying defects of all of these mutants (Trentet al. 1983; Thomaset al. 1993; this work). Based on these findings, we propose that egl-4 relays sensory cues to the egg-laying circuit through the activity of the TGF-β pathway. This modulation may occur via direct or indirect regulation of the flp-1-encoded neuropeptides, or these genes could act in an independent pathway. Although egl-4 mutations are pleiotropic, it is unlikely that egl-4 affects some very general aspect of neuronal function. Other egg-laying mutants are not Daf-c and their phenotypes are not suppressed by daf-3 and daf-5. Thus, egl-4 perturbs a specific aspect of neuronal function.
Where does egl-4 act? It is interesting to note that egl-4 mutants show specific chemosensory defects. egl-4 shows strong defects in sensing diacetyl, but responds relatively well to pyrazine and trimethylthiazole, chemicals also sensed by the AWA neurons. Based on the numerous pleiotropies of egl-4 mutants, it is unlikely that egl-4 functions only in the primary chemosensory responses. It has been shown that animals adapted to one odorant continue to respond to other odorants sensed by the same neuron type (Colbert and Bargmann 1995). This could occur if, for instance, responses to different odorants resulted in the activation of distinct signaling pathways downstream of the sensory neuron. For example, the ASH sensory neurons use a glutamate-mediated pathway to respond to one type of stimulus, while other stimuli sensed by the same neurons appear to use a distinct mechanism (Hartet al. 1995; Maricqet al. 1995). It is possible that egl-4 functions similarly to mediate specific odorant response pathways. This would suggest that a site of egl-4 function could be interneurons. This is also consistent with its role in regulating egg laying, since flp-1 is known to be expressed in interneurons that integrate sensory signals (Schinkmann and Li 1992). We expect that elucidation of the molecular nature of egl-4 and its sites of action will reveal further information regarding its role in regulating development and behavior in C. elegans.
Acknowledgments
We thank Cori Bargmann in whose lab this work was initiated. We are grateful to Elizabeth Malone Link for advice and suggestions during the course of this work. We thank Don Riddle, David Miller, and Isao Katsura for permission to cite unpublished work; Bob Horvitz, Don Riddle, Chris Li, Gary Ruvkun, and the Caenorhabditis Genetics Center for providing us with strains; and the Sengupta lab for advice and ideas. We also thank Elizabeth Malone Link and Cori Bargmann for critical comments on the manuscript. This work was supported by National Institutes of Health grants GM56223 (P.S.) and GM48700 (J.T.), and funds from the Searle Scholar Foundation and Packard Foundation (P.S.). M.A. was supported by a Howard Hughes Medical Institute Predoctoral Fellowship.
Footnotes
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Communicating editor: R. K. Herman
- Received April 6, 2000.
- Accepted May 15, 2000.
- Copyright © 2000 by the Genetics Society of America
