Dauer Formation Induced by High Temperatures in Caenorhabditis elegans

Corresponding author: James H. Thomas, Department of Genetics, University of Washington, Box 357360, Seattle, WA 98195., jht{at}genetics.washington.edu (E-mail)

Communicating editor: P. ANDERSON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Dauer formation in Caenorhabditis elegans is regulated by several environmental stimuli, including a pheromone and temperature. Dauer formation is moderately induced as the growth temperature increases from 15° to 25°. Here we show that dauer formation is very strongly induced at a temperature of 27° in both wild-type animals and mutants such as unc-64, unc-31, and unc-3, which do not form dauers at 25°. A 27° temperature stimulus is sufficient to induce dauer formation in wild-type animals independent of pheromone. Analysis of previously described dauer mutants at 27° reveals a number of surprising results. Several classes of mutants (dyf, daf-3, tax-4, and tax-2) that are defective in dauer formation at lower temperatures reverse their phenotypes at 27° and form dauers constitutively. Epistasis experiments place unc-64 and unc-31 at a different position in the dauer pathway from unc-3. We also uncover new branches of the dauer pathway at 27° that are not detected at 25°. We show that epistatic gene interactions can show both quantitative and qualitative differences depending on environmental conditions. Finally, we discuss some of the possible ecological implications of dauer induction by high temperatures.

UNDER favorable environmental conditions, the nematode Caenorhabditis elegans life cycle consists of four larval stages (L1–L4) in the progression to an adult. However, if environmental conditions are unfavorable, a worm may arrest development following the L2 stage and become a dauer larva. Dauers have several morphological and physiological alterations that make them well adapted for long-term survival and resistant to harsh environmental conditions (CASSADA and RUSSELL 1975 ; RIDDLE and ALBERT 1997 ). Upon the return of favorable environmental conditions, dauers can recover and complete normal development. Since environmental conditions outside of the laboratory presumably are frequently unfavorable, correct regulation of dauer formation is likely to be of considerable ecological importance.

Three environmental cues are known to regulate the decision to form a dauer. The most critical is the concentration of a pheromone that is constitutively secreted throughout the life cycle, serving as an indicator of population density (GOLDEN and RIDDLE 1982 ; OHBA and ISHIBASHI 1982 ). The pheromone has been partially purified and consists of several related molecules similar to hydroxylated fatty acids (GOLDEN and RIDDLE 1984C ). Temperature and food signals modulate the dauer decision, with higher temperatures and lower amounts of food increasing the frequency of dauer formation (GOLDEN and RIDDLE 1984A , GOLDEN and RIDDLE 1984B ). However, it has been thought that pheromone is both necessary and sufficient for dauer formation. The fact that pheromone is capable of inducing dauer formation at low temperatures in the presence of ample food suggests that it is sufficient to induce dauer formation. Evidence for the necessity of pheromone comes from analysis of the daf-22 mutant, which does not produce pheromone (GOLDEN and RIDDLE 1985 ). daf-22 mutants do not form dauers if crowded and starved (while wild-type worms do) and a pheromone extract prepared from daf-22 mutants is not capable of inducing dauer formation in wild-type animals. Furthermore, daf-22 mutants are capable of forming dauers in response to exogenously supplied pheromone.

Pheromone is sensed by chemosensory neurons that have endings directly exposed to the environment in the bilateral amphid organs at the tip of the worm's nose (PERKINS et al. 1986 ). By killing cells with a laser, researchers have shown that several different amphid neurons regulate pheromone response (BARGMANN and HORVITZ 1991 ; SCHACKWITZ et al. 1996 ). The ASI and ADF neurons repress dauer formation in the absence of pheromone and derepress dauer formation in the presence of pheromone. Killing these cells leads to inappropriate dauer formation. In contrast, the ASJ neuron promotes dauer formation in the presence of pheromone. Killing this cell leads to reduced responsiveness to pheromone. All the other amphid sensory neurons have been killed with little documented effect on dauer formation.

Genetic analysis of dauer formation has led to the isolation of many mutants that fall into two general classes: dauer formation constitutive (Daf-c) mutants form dauers inappropriately under noninducing conditions while dauer formation defective (Daf-d) mutants fail to form dauers under inducing conditions. Analysis of synergistic and epistatic gene interactions in many double mutants has led to the formal genetic pathway shown in Fig 1A (VOWELS and THOMAS 1992 ; THOMAS et al. 1993 ; GOTTLIEB and RUVKUN 1994 ). The parallel branches of the genetic pathway have been correlated to specific sensory neurons acting in parallel (SCHACKWITZ et al. 1996 ). Killing ASJ suppresses the Daf-c phenotype of daf-11 and daf-21 mutants but has little effect on the group II Daf-c mutants, suggesting that the group I Daf-c pathway functions through this neuron. Cell isolation experiments suggest that ASI and ADF mediate the group II Daf-c pathway. Killing all the other amphid neurons in daf-7 or daf-1 mutants did not prevent dauer formation, suggesting that these neurons were sufficient to convey the Daf-c signal. Furthermore, a daf-7::gfp construct showed expression only in ASI (REN et al. 1996 ; SCHACKWITZ et al. 1996 ).

View larger version (29K): In this window In a new window Download PPT slide   Figure 1. Genetic pathways that regulate dauer formation. (A) The pathway as determined at 25°. (B) Additions to the pathway as observed at 27°. Genes with different phenotypes at 27° (dyf and daf-3) are boxed and additional branches to the pathway are drawn with dashed lines. unc-64, unc-31, and unc-3 are added to the pathway. Since unc-3 acts partially in parallel to the group II Daf-c genes, it is drawn in parentheses. See text for detailed explanations.

Mutations in the large group of Daf-d genes (dyf) located downstream of the group I Daf-c genes and upstream of the group II Daf-c genes affect the structure of the ciliated sensory endings of the amphid neurons, rendering them nonresponsive to pheromone (PERKINS et al. 1986 ; VOWELS and THOMAS 1994 ; STARICH et al. 1995 ). Mutants of this class (known as cilium-structure mutants) can easily be scored by their inability to take up the fluorescent dye FITC in their amphid neurons (PERKINS et al. 1986 ), a phenotype known as dye-filling defective (Dyf). Since these mutations suppress the daf-11 and daf-21 Daf-c phenotypes, it is thought that daf-11 and daf-21 function in the sensory endings. The daf-11 gene has been shown to encode a transmembrane guanylyl cyclase (BIRNBY et al. 2000 ), consistent with a role in sensory transduction. The group II genes have been shown to encode components of a TGF-ß signaling pathway (GEORGI et al. 1990 ; ESTEVEZ et al. 1993 ; REN et al. 1996 ; PATTERSON et al. 1997 ; INOUE and THOMAS 2000 ). Such pathways have usually been implicated as functioning in development (KINGSLEY 1994 ), so the involvement in neuronal function is unexpected. How these molecules might contribute or respond to neuronal activity is not understood, but there is evidence that daf-7 gene expression is affected by the pheromone, temperature, and food signals that regulate dauer formation (REN et al. 1996 ; SCHACKWITZ et al. 1996 ). The third branch of the pathway (the insulin branch) consists of genes encoding components of an insulin receptor signaling pathway (MORRIS et al. 1996 ; KIMURA et al. 1997 ; LIN et al. 1997 ; OGG et al. 1997 ; PARADIS and RUVKUN 1998 ; PARADIS et al. 1999 ). It is not yet clear whether the activity of this pathway is regulated by sensory input. The final gene in the pathway daf-12 encodes a steroid hormone receptor (YEH 1991 ) and likely mediates the execution of the dauer developmental program in response to the neuronal inputs.

While much progress has been made in identifying the molecular and cellular components involved in regulating dauer formation, there is still much to be learned. For example, it is not known what cells sense temperature and food, nor at what step or branch of the genetic pathway these signals are integrated. Furthermore, while many screens have been done for genes with a strong Daf-c phenotype at 25°, there is evidence that many other genes have roles in regulating dauer formation (AVERY 1993 ; KATSURA et al. 1994 ; MALONE et al. 1996 ; IWASAKI et al. 1997 ; PRASAD et al. 1998 ; TAKE-UCHI et al. 1998 ; AILION et al. 1999 ; KOGA et al. 1999 ; SZE et al. 2000 ). Many of these genes have a synthetic Daf-c (Syn-Daf) phenotype at 25° that requires mutations in two genes to generate a detectable Daf-c phenotype, explaining why they had been missed in screens at 25°. Here we show that single mutants of several of these genes have highly penetrant Daf-c phenotypes at 27°, namely unc-3, which encodes a transcription factor (PRASAD et al. 1998 ), and unc-31 and unc-64, which encode homologs of CAPS and syntaxin, proteins that regulate secretion and synaptic transmission (LIVINGSTONE 1991 ; ANN et al. 1997 ; OGAWA et al. 1998 ; SAIFEE et al. 1998 ). We characterize the wild-type response to 27° and show that dauer formation is strongly induced at this temperature in a pheromone-independent manner. We perform epistasis experiments on the new genes to place them in the dauer pathway. This study reveals new branches to the dauer pathway that are not detected at 25° and demonstrates that a number of genes unexpectedly have both positive and negative regulatory influences on dauer formation. Fig 1B illustrates some of the differences in the dauer pathway observed at 27°.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

General growth conditions and strain maintenance:
C. elegans strains were cultured and manipulated using standard methods (BRENNER 1974 ). All strains were derivatives of the Bristol wild-type strain N2. Worms were grown on Escherichia coli strain TJ2, a derivative of OP50. TJ2 has always been used as the standard E. coli strain in this lab. TJ2 shows auxotrophic differences from OP50 (data not shown). The possible effect of these differences on C. elegans dauer formation was not investigated. TJ2 was cultured by serial passage for up to a few months before returning to the original frozen culture. This article follows the standard C. elegans nomenclature (HORVITZ et al. 1979 ). The CB246 unc-64(e246) strain was found to have an unlinked temperature-sensitive sterile mutation. unc-64(e246) was outcrossed to remove this mutation and all assays were performed on the outcrossed strain. A complete list of strains and mutations used is available upon request.

Dauer formation assays:
Parents raised continuously on food at 20° were allowed to lay eggs for 3–6 hr at room temperature (22°) and progeny were incubated at the assay temperature. Dauer and nondauer animals were counted after 100 hr at 15°, 65 hr at 20°, 54 hr at 22°, 48 hr at 25°, and 44 hr at 27°, which permitted correct scoring of transient dauers that recover rapidly. Dauer assays have a tendency to show quantitative variability from experiment to experiment (presumably due to the input of multiple environmental conditions that are hard to control rigorously), and this was especially true at 27° due to the particular sensitivity of dauer formation around this temperature. Temperature differences of 0.5° or less can have significant quantitative effects on dauer formation at temperatures near 27°. We found that there was temperature variability of at least 0.5° both at different locations within an incubator and at the same location of an incubator examined at different times. To demonstrate that such variability could have significant effects on dauer formation, we performed an experiment in which we assayed unc-31(e928) dauer formation on many plates distributed throughout our incubator. Spatial differences in temperature ranged from 26.5° to 27.1° and unc-31 ranged from 60 to 100% dauers in agreement with the local temperature. Because of such spatial and temporal variability in dauer formation, each table in this article presents the results from a single experiment in which all strains were assayed in parallel in close proximity in the incubator. In cases where a table is divided by extra space, each section of the table presents the results from a single experiment, but different sections represent different experiments. Experiments were repeated multiple times with quantitative variability in the absolute numbers, but the relative differences between strains were consistent. For assays at 25° and 27°, temperature was measured using a thermometer (ASTM no. 23C from VWR) accurate to 0.1°. This thermometer was placed in close proximity to the assay plates on the same shelf of the incubator. The reported temperature for any given experiment is an average of the temperature measured at the start of the experiment when plates were placed at the assay temperature and the end of the experiment when plates were removed to count dauers. However, since there is temporal variability, this reported temperature might not represent the average temperature of the assay. Temperature in the text is referred to as 25° or 27° for simplicity, but in actuality "25°" was 25.0°–25.6° and "27°" was 26.6°–27.1°. The temperature on the surface of the agar was not measured, so the temperature experienced by the worms may vary slightly from the measured temperature. The primary 27° incubator was a heated incubator placed in a room at 4°. A small fan was placed on the top shelf of the incubator to minimize temperature variability within the incubator. Experiments performed at 27° in a heating/refrigerating incubator at room temperature or in a sealed plastic tupperware container submerged in a 27° water bath gave similar results.

Assays of dauer formation at 27° present technical problems in addition to the variability described above. Some strains (e.g., N2) form dauers at 27°, which recover within a few hours. Tightly synchronized egg lays could not solve this problem completely, since growth of strains at 27° tends to be somewhat asynchronous, even when egg lays were synchronous. This is probably due to the general unhealthiness of worms grown at high temperatures. In all dauer formation assays, animals at the L1 or L2 stage of development were counted, but not included in the presented data.

Pheromone assays:
Plates with partially purified dauer pheromone were prepared as described (VOWELS and THOMAS 1994 ). Different pheromone preparations were used in different experiments. Dauer formation is induced slightly on pheromone assay media (without pheromone) relative to standard nematode growth plates. Within an experiment, all strains were grown in duplicate at each pheromone concentration and plates were randomly distributed in a sealed plastic tupperware container with a moist paper towel to prevent drying of the small plates. Dauer formation is partially suppressed by drying of the plate (data not shown).

Starvation assays:
Dauer formation in response to starvation was assayed by picking two adult animals to plates at 20° and checking to see when the bacterial lawn was completely gone. Four days later the plates were flooded with 1% SDS and scored after 15 min for the presence of dauers (live thrashing animals).

Construction of double and triple mutant strains:
Double and triple mutant strains were constructed and confirmed by the methods described previously (VOWELS and THOMAS 1992 ; THOMAS et al. 1993 ). A detailed description of strain constructions is available upon request.

Dominance tests:
Dominance of Daf-c mutants at 27° was assayed by mating wild-type males to marked daf-c strains at 20° for 1 day, then performing synchronous egg lays at room temperature and allowing the broods to develop at 27°. Unmarked dauers and nondauers were counted. For daf-7, the cross was also performed in the reciprocal direction, mating heterozygous daf-7/+ males to unc-33(e204) hermaphrodites, to control for the possibility of a maternal effect.

Expression of daf-7::gfp:
Animals carrying the integrated daf-7::gfp array saIs8 were grown at various temperatures to the L2 stage at which maximal expression was observed (SCHACKWITZ et al. 1996 ). Green fluorescent protein (GFP) fluorescence was observed at 1000x magnification using a compound microscope with UV illumination. ASI was identified by cell position viewed with Nomarski optics.

Cell kills:
ASI and ADF were identified by cell position and killed by a laser in L1 larvae within 2 hr of hatching as described (AVERY and HORVITZ 1987 ; SCHACKWITZ et al. 1996 ), except that parents were grown at 20° rather than being preadapted to the assay temperature.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Synthetic Daf-c genes:
Screens for simple loss-of-function mutants with a strong Daf-c phenotype at 25° have probably been saturated (MALONE and THOMAS 1994 ). However, a number of mutants with Syn-Daf phenotypes have been described in recent years (AVERY 1993 ; KATSURA et al. 1994 ; IWASAKI et al. 1997 ; TAKE-UCHI et al. 1998 ; AILION et al. 1999 ; DANIELS et al. 2000 ). In this article, we consider three of these genes: unc-3, unc-31, and unc-64. As shown in Table 1, the unc-64; unc-31, unc-64; unc-3, and unc-31; unc-3 double mutant strains are strongly Daf-c at 25°, while the three single mutants are either not Daf-c or are only weakly Daf-c (unc-3 appears to be particularly variable at 25°, possibly due to local starvation of part of a plate). The Syn-Daf phenotypes are less penetrant at 15°, reflecting the intrinsic temperature sensitivity of dauer formation (GOLDEN and RIDDLE 1984B ; MALONE and THOMAS 1994 ).

We examined the phenotypes of several triple mutants of two Syn-Daf genes with a Daf-d gene in order to place the Syn-Daf mutant in the dauer pathway (see Fig 1). The Daf-c phenotype of an unc-64; unc-31 double mutant was not suppressed by either daf-3 or daf-5 but was completely suppressed by daf-12. The unc-31; unc-3 double mutant was suppressed by daf-5 at 15° but not at 25° and was suppressed by daf-12 at both temperatures. These results suggest that the Syn-Daf combinations act genetically in parallel to or downstream of the group II pathway shown in Fig 1. In support of this idea, the unc-64; unc-31 double mutant was completely suppressed by mutations in daf-16 (data not shown). The partial suppression of unc-31; unc-3 by daf-5 is consistent with the idea that unc-3 acts in the group II pathway (see below).

A synthetic Daf-c phenotype could result from true genetic redundancy or from the additive effect of several weak Daf-c phenotypes. To test whether the single mutants are shifted toward forming dauers, we measured dauer formation in response to various amounts of exogenous pheromone. As shown in Fig 2A, unc-3, unc-31, and unc-64 mutants are all hypersensitive to dauer pheromone at 25°. The Syn-Daf mutant aex-3 is not hypersensitive to dauer pheromone (data not shown), indicating that pheromone hypersensitivity is not a property of all Syn-Daf mutants. unc-3 and unc-64 mutants remain hypersensitive to dauer pheromone when assayed at 22°, but the unc-31(e928) mutant at 22° is actually less sensitive to pheromone than N2 (Fig 2B). To determine whether this surprising phenotype is specific to the e928 allele (a deletion of most of the unc-31 gene and expected null; LIVINGSTONE 1991 ), we assayed two other alleles of unc-31 for pheromone response at 22° and 25°. All three alleles exhibited clear hypersensitivity at 25° (Fig 2C) and reduced sensitivity at 22° (Fig 2D), indicating that this phenotype is not allele specific. While the reversal of the unc-31 response is not easy to interpret (see DISCUSSION), the hypersensitivity of unc-3, unc-31, and unc-64 at 25° indicates that some single Syn-Daf mutations do affect dauer formation on their own. This observation is extended in the following section.

View larger version (26K): In this window In a new window Download PPT slide   Figure 2. Dauer formation of unc-64, unc-31, and unc-3 mutants in response to exogenous pheromone. Each graph plots the percentage of animals that formed dauers in response to different concentrations of pheromone at the given temperature. Approximately 100–200 animals were counted at each concentration of pheromone.

Syn-Daf single mutants are Daf-c at 27°:
During our study of Syn-Daf mutants, we made a fortuitous discovery while performing experiments in which the incubator temperature was accidentally set slightly high, at approximately 27°. At this temperature, we found that unc-3, unc-31, and unc-64 mutants had strong Daf-c phenotypes on their own (Table 2). The Daf-c phenotype of these mutants was clearly weaker at 26°, indicative of the strong temperature dependence. Wild-type N2 worms did not form dauers in initial experiments at 27°. However, during many repetitions of this experiment, we noticed occasional dauers on N2 plates. It is now clear that N2 is weakly Daf-c at 27°, but formation of dauers is variable from experiment to experiment, probably due to slight differences in incubation temperature (see MATERIALS AND METHODS). Furthermore, N2 dauers formed at 27° recover rapidly at 27° (data not shown), which can make scoring difficult, even in synchronized broods. The strong 27° Daf-c phenotype is called the high temperature-induced dauer formation (Hid) phenotype to distinguish it from the weak 27° Daf-c phenotype of wild type. N2 generally has <20% dauers at temperatures around 27°, but on rare occasions was seen to make up to 75% dauers. The Hid phenotype of unc-3, unc-31, and unc-64 was not allele specific. unc-3(e54), unc-3(e95), unc-3(cn4146), unc-31(u280), unc-31(e169), unc-64(md1259), and unc-64(md130) were all found to have a Hid phenotype (AILION et al. 1999 and data not shown). The Hid phenotype of unc-3 mutants was confirmed by others subsequent to our finding (PRASAD et al. 1998 ).

Dauers formed at 27° in mutant or wild-type strains are often paler than dauers of the same strains formed at 25°. To assess whether 27° dauers are true dauers (as opposed to partial dauers such as those made by daf-16 mutants), we scored several dauer-specific features that can be visualized by Nomarski microscopy: presence of dauer alae, remodeling of the pharynx, presence of hypodermal bodies, and the presence of highly refractile material in the gut (VOWELS and THOMAS 1992 ). We also scored another dauer feature, resistance to 1% SDS. N2, unc-3, unc-31, and unc-64 dauers formed at 27° had all the characteristic features of dauers formed at lower temperatures and thus are indeed true dauers (though there were often fewer hypodermal bodies in 27° dauers). The amount of refractile material in the gut correlated with the darkness of a dauer seen using a dissecting microscope.

Temperature sensitivity of pheromone response:
The unc-3, unc-31, and unc-64 mutants are clearly sensitive to small temperature differences in the narrow range from 25° to 27°. To see if this sensitivity is specific to these mutants or is a wild-type phenomenon, we assayed N2 dauer formation in response to exogenous pheromone at various temperatures. As shown in Fig 3A, temperature had a modest effect on wild-type pheromone response from 15° to 25° as shown previously (GOLDEN and RIDDLE 1984A , GOLDEN and RIDDLE 1984B ). However, N2 responded much more strongly to pheromone at 27° than at 25°, suggesting that wild-type dauer formation is highly sensitive to this temperature difference and that the mutant phenotypes are likely to reflect an underlying wild-type sensitivity.

View larger version (17K): In this window In a new window Download PPT slide   Figure 3. Dauer formation of N2 and the ttx-1 mutant in response to exogenous pheromone at different temperatures. Approximately 100–200 animals were counted at each concentration of pheromone.

We also performed similar pheromone response assays on the mutant ttx-1(p767), which has defects in the morphology of the candidate thermosensory cell AFD and defects in thermotaxis behavior (HEDGECOCK and RUSSELL 1975 ; PERKINS et al. 1986 ; MORI and OHSHIMA 1995 ). As shown in Fig 3B, the ttx-1 mutant formed more dauers as the temperature was increased from 15° to 25° and like N2 showed an extremely strong response at 27°, including a low frequency of dauer formation in the absence of exogenous pheromone. Thus, it seems unlikely that AFD is solely responsible for the temperature input to dauer formation. As demonstrated before (GOLDEN and RIDDLE 1984B ), the ttx-1 mutant is hypersensitive to the dauer pheromone, suggesting that AFD plays some role in dauer formation, albeit not an essential one.

Dauer formation at high temperatures can occur independently of pheromone:
As noted earlier, N2 dauer formation at 27° is much more sensitive to pheromone than at 25°. However, N2 also forms a low frequency of dauers at 27° on plates with ample food and no exogenously added pheromone, which does not happen at 25°. Two possibilities could account for this phenomenon. Dauer formation by N2 at 27° could result from endogenous pheromone made by the tested larvae, but present at a level insufficient to induce dauer formation at 25°. Alternatively, dauer formation at 27° could occur independently of pheromone. To distinguish between these possibilities, we assayed dauer formation of daf-22(m130) mutant animals at 27°. The daf-22 mutant does not produce pheromone and has a Daf-d phenotype at lower temperatures that can be rescued by exogenously supplied pheromone (GOLDEN and RIDDLE 1985 ). When grown at 27°, daf-22 mutants formed dauers at a frequency similar to N2. daf-22 dauers formed at 27° were examined by Nomarski microscopy and had all the features typical of dauers. Thus, it appears that in addition to a highly sensitized pheromone response, dauer formation can also occur independently of pheromone at 27°, although the possibility that daf-22 animals make pheromone only at 27° is not excluded.

Dauer formation at 27° in Daf-d mutants:
The finding that daf-22, a Daf-d mutant, behaves similarly to N2 at 27° in producing dauers led us to examine other Daf-d mutants at 27°. Daf-d mutants are characterized by several phenotypes at 25° or lower temperatures: inability to form dauers following starvation, inability to form dauers in response to exogenously added pheromone, and suppression of Daf-c mutants upstream in the dauer pathway. Since dauer formation at 27° can occur independently of pheromone, these phenotypes of Daf-d mutants are not necessarily predicted to be the same at 27°.

As shown in Table 3, Daf-d mutants show several unexpected phenotypes at 27°. Mutations in the Dyf genes such as daf-10 and osm-6, which affect the structure of the ciliated endings of the amphid sensory neurons, lead to a Daf-c phenotype at 27°. This varies in strength from gene to gene but, in the strongest (e.g., osm-6, osm-5, che-11), is almost completely penetrant and is always significantly stronger than N2. This Hid phenotype was seen in all 16 Dyf mutants that we tested (Table 4) and was confirmed by others subsequent to our finding (APFELD and KENYON 1999 ). The daf-6(e1377) mutation, which affects the structure of the amphid sheath cell (ALBERT et al. 1981 ), did not affect dauer formation as strongly. In multiple assays of the daf-6 mutant, a weak Daf-c phenotype at 27° was occasionally seen but usually it formed dauers at a level similar to N2. However, unlike N2 and like the other Dyf mutants, daf-6 dauers failed to recover at 27°, consistent with a defect in responding to dauer recovery conditions. Another mutant of interest is che-12(e1812), which has defects in secretion of matrix material by the amphid sheath cell but is not strongly defective in dye filling by the amphid sensory neurons (PERKINS et al. 1986 ). At 27°, che-12 mutants formed dauers at a level similar to N2 (Table 3) and the dauers recovered efficiently. Similarly, the mec-1(e1066) and mec-8(e398) mutants, which have defects in the fasciculation of the amphid cilia (LEWIS and HODGKIN 1977 ; PERKINS et al. 1986 ), are not Daf-d at lower temperatures and are not Hid (data not shown). Thus, the Hid phenotype appears to be specific to mutants with defects in the structure of the ciliated neurons themselves. Mutants that affect the structure of the amphid pore in other ways are not Hid.

Mutations in either daf-3 or daf-5 exhibit a strong Daf-d phenotype at 25° or lower temperatures and strongly suppress the Daf-c phenotype of group II Daf-c mutations. Surprisingly, daf-3 mutants were strongly Daf-c at 27° while daf-5 mutants behaved similarly to N2, forming dauers at a low percentage (Table 3). Since daf-3 and daf-5 had indistinguishable phenotypes in other assays, we tested whether these 27° phenotypes were allele specific. Eleven alleles of daf-3, including mgDf90, a deletion of the entire daf-3 coding sequence (PATTERSON et al. 1997 ), exhibited the Hid phenotype while all four daf-5 alleles tested behaved like N2 (Table 4), indicating that these phenotypes are not allele specific. Thus, although daf-3 and daf-5 have indistinguishable phenotypes at 25°, they have strikingly different phenotypes at 27°.

Mutations in the daf-16 gene suppress the Daf-c phenotype of mutants in the insulin branch of the dauer pathway. At 27°, daf-16(m27) mutants formed partial dauers at a low frequency similar to that of N2 dauer formation (Table 3, Table 5, and Table 11). This result was seen in several other daf-16 alleles, including m26 and mgDf50, a deletion of almost all of the daf-16 coding sequence (OGG et al. 1997 ). Thus, dauer formation at 27° can occur independently of the insulin pathway or of the group I or group II Daf-c signaling pathways. However, dauer formation at 27° depends absolutely on the daf-12 gene (Table 3), indicating that dauer induction at 27° shares a common output with dauer induction by other stimuli. The phenotypes of the tested Daf-d mutants at 25° and 27° are summarized in Table 4.

Response of Daf-d mutants to pheromone at 27°:
Daf-d mutants do not respond to pheromone or respond only very weakly at temperatures at or below 25°. The observation that all Daf-d mutants except daf-12 were capable of dauer formation at 27° led us to examine whether these mutants responded to pheromone at 27°. As shown in Table 5, N2 responded strongly to pheromone at a temperature near 25° while the osm-6 and daf-12 mutants did not respond at all and daf-3 and daf-5 mutants responded only very weakly. daf-16 responded to a lesser degree than N2 and made partial dauers. At 27°, N2 still responded strongly and daf-3 and daf-5 mutants responded strongly as well. osm-6 and daf-12 still failed to respond and daf-16 continued to respond to a lesser extent. Assaying the pheromone responsiveness of daf-3 and osm-6 at 27° was complicated by the fact that these mutants are Daf-c without pheromone at 27°. To circumvent this problem, we assayed pheromone responsiveness at a slightly lower temperature at which the Daf-c phenotypes of daf-3 and osm-6 were only partially penetrant. Under these conditions, daf-3 responded strongly to pheromone while osm-6 (and several other Dyf mutants) did not respond at all (data not shown). Thus, pheromone-induced dauer formation at 27° depends on the ciliated endings of sensory neurons, as at lower temperatures, but does not depend on the activities of the daf-3 and daf-5 genes.

Dauer formation at 25° and 27° in tax-4 and tax-2 mutants:
To continue our characterization of dauer mutants at 27°, we examined mutants in the genes tax-4 and tax-2. tax-4 and tax-2 encode - and ß-subunits of a cyclic nucleotide-gated (CNG) ion channel that appears to be part of the signal transduction machinery in the amphid cilia (COBURN and BARGMANN 1996 ; KOMATSU et al. 1996 ). tax-4 and tax-2 mutants have interesting dauer phenotypes at 25° that appear to be a combination of dauer-promoting and dauer-repressing activities; i.e., mutations in tax-4 or tax-2 suppress the Daf-c phenotype of mutants in the group I pathway but enhance the Daf-c phenotype of mutants in the group II pathway (COBURN et al. 1998 ). This is suggestive of a tax-4/tax-2 site of action in the group I pathway. To investigate this further, we examined the phenotype of tax-4(ks11) double mutants with Daf-d genes. tax-4(ks11) has a strong Daf-c phenotype at 25° (Table 6) so epistasis could be performed at this temperature. Other tax-4 mutants including a putative null (p678) are only weakly Daf-c at 25°. Mutations in the group I cilium-structure Daf-d gene osm-3 or in daf-12 completely suppressed the Daf-c phenotype of tax-4(ks11), while mutations in daf-5 and daf-16 only partially suppressed it (Table 6). This is very similar to epistasis data seen with the group I Daf-c gene daf-11 (VOWELS and THOMAS 1992 ), providing further evidence that tax-4 functions in the group I pathway.

At 27°, all tax-4 alleles exhibited a strong Daf-c phenotype and the dauers did not recover (Table 7). The tax-2(p691) mutant was also strongly Daf-c at 27° and failed to recover. The p691 mutation affects the same proline residue in the channel pore as the strongest Daf-c tax-4 allele ks11 (COBURN and BARGMANN 1996 ; KOMATSU et al. 1996 ). The tax-2(p671) mutant was moderately Daf-c at 27°, but the dauers recovered efficiently. The p671 mutation may simply be a weaker mutation of tax-2 than p691, with a smaller defect in both dauer formation and dauer recovery. The tax-2(p694) mutant was not Daf-c at 27°. As inferred from an analogous GFP expression construct, this mutation eliminates expression of the channel in the AFD, ASE, ADE, and BAG neurons but does not affect expression or function of the channel in seven other cells (COBURN and BARGMANN 1996 ), suggesting that the Hid phenotype of the other alleles results from loss or impaired channel function in other cells.

The native CNG channel is likely to be a heteromer formed of both TAX-4 -subunits and TAX-2 ß-subunits. However, the TAX-4 protein may be able to form a functional homomeric channel in the absence of TAX-2 although the reverse is unlikely (KOMATSU et al. 1996 , KOMATSU et al. 1999 ). To determine whether tax-4 phenotypes depended on tax-2 or vice versa, we examined dauer formation in tax-2; tax-4 double mutants. As shown in Table 8, all three tax-2 mutations suppressed the Daf-c phenotype of tax-4(ks11) at 25°, with suppression stronger by the p691 and p694 mutations, consistent with the idea that p671 is a weaker mutation. The suppression by tax-2(p694) implies that the 25° Daf-c phenotype of tax-4(ks11) depends on TAX-2 function in AFD, ASE, ADE, or BAG. However, neither tax-4(ks11) nor tax-4(p678) was suppressed by any of the tax-2 mutations at 27°, suggesting that the 27° Daf-c phenotype of tax-4 mutations does not depend on TAX-2 function in AFD, ASE, ADE, or BAG. The dauer recovery defect of tax-4 mutants at 27° was partially suppressed by the tax-2 mutations p691 and p694, implying that the dauer recovery phenotype of tax-4 depends on TAX-2 function in AFD, ASE, ADE, or BAG. The fact that tax-2 mutations have effects in a putative tax-4 null background also suggests that the TAX-2 protein can function in the absence of TAX-4, possibly as the partner of other -subunits.

Responses of tax-4 and tax-2 mutants to pheromone:
To further examine the role of tax-4 and tax-2 in dauer formation, we assayed dauer formation of tax-4 and tax-2 single and double mutants in response to exogenous pheromone. We performed these assays at both 25° and 22° since the tax-4(ks11) mutant is strongly Daf-c at 25° without pheromone and because there was a precedent for opposite pheromone responses at these two temperatures (unc-31, see above). As shown in Table 9, the three tax-4 mutants have a weak pheromone response and the three tax-2 mutants do not respond to pheromone at all. The complete pheromone insensitivity of the tax-2(p694) mutant is particularly notable as it suggests that this defect is due to a site of action in one or more of the AFD, ASE, ADE, or BAG neurons, none of which have been implicated previously in regulating the response to pheromone. The pheromone responsiveness of tax-4 mutants appears to be suppressed by tax-2(p691) but not by tax-2(p671), though the weakness of pheromone induction of dauer formation in tax-4 single mutants makes this somewhat difficult to interpret. Dauer formation of tax-4(p678) in the absence of pheromone was strongly suppressed by tax-2(p691) and partially suppressed by tax-2(p694), again suggesting that the TAX-2 protein may function in the absence of TAX-4.

The lack of pheromone responsiveness of tax-2 mutants and reduced response of tax-4 led us to examine these mutants for defects in dye-filling of the amphid sensory neurons, a phenotype characteristic of cilium-structure mutants (PERKINS et al. 1986 ; STARICH et al. 1995 ) that also fail to respond to pheromone. Six amphid neuron pairs (ASJ, ADF, ASH, ASI, ADL, and ASK) fill with the fluorescent dye FITC (HEDGECOCK et al. 1985 ). tax-4 and tax-2 mutants were capable of FITC dye-filling by all six cells, though filling of ASJ and ASI was often weaker or not detectable (data not shown). This could indicate a weak dye-filling defect specific to these cells, but since ASJ and ASI fill more weakly in wild type this could also simply reflect a general weak defect that is only detectable in these cells. COBURN and BARGMANN 1996 showed that both ASI and ASJ fill relatively normally with the dye DiO in tax-2 and tax-4 mutants. Thus, tax-4 and tax-2 mutants do not appear to have strong defects in the structure of the amphid cilia.

Epistasis based on the Hid phenotype:
At least three parallel pathways regulate dauer formation (Fig 1). These pathways were inferred by examining epistatic interactions among Daf-c and Daf-d genes at temperatures ranging from 15° to 25° (VOWELS and THOMAS 1992 ; THOMAS et al. 1993 ; GOTTLIEB and RUVKUN 1994 ). To determine the pathway in which the Hid mutants function, we built double mutants between Hid mutants and Daf-d mutants in each branch of the pathway. We also reexamined epistasis of the previously characterized Daf-c genes to test whether the same epistatic relationships hold at 27° as at lower temperatures.

Double mutants with daf-22: As shown above, C. elegans is capable of weak pheromone-independent dauer formation at 27° but is also highly sensitized to pheromone at 27°. Since several Hid mutants are hypersensitive to pheromone, it was possible that the Hid phenotype was caused by an increased response to low levels of endogenous pheromone that only weakly induced dauer formation of wild type. To determine whether any Hid phenotypes depend on pheromone, we built double mutants of Hid mutants with daf-22, which does not make pheromone. daf-22 double mutants with unc-3(e151), unc-31(e928), unc-64(e246), osm-6(p811), and daf-3(sa213) formed 100% dauers at 27°, indicating that the Hid phenotype does not depend on endogenous pheromone production.

Double mutants with dyf genes: Mutations in many Dyf genes suppress the Daf-c phenotype of group I Daf-c mutants at 25° (VOWELS and THOMAS 1992 ; STARICH et al. 1995 ). Epistasis with the Dyf mutants at 27° is complicated by the fact that Dyf mutants are Daf-c on their own at 27°. Nevertheless, we built a number of double mutants of unc-3, unc-31, and unc-64 with mutations in the Dyf genes osm-1, osm-3, osm-5, osm-6, che-3, che-11, and daf-10. Surprisingly, all such double mutants had a Syn-Daf phenotype at temperatures from 15° to 25° (data not shown). This interaction suggests that unc-3, unc-31, and unc-64 act in parallel to the group I pathway. This also suggests that Dyf mutations have both positive and negative effects on dauer formation between 15° and 25°.

Double mutants with daf-3 and daf-5: Mutations in daf-3 and daf-5 completely suppress the Daf-c phenotype of group II Daf-c mutants at 25° and partially suppress the Daf-c phenotype of group I Daf-c mutants (VOWELS and THOMAS 1992 ; THOMAS et al. 1993 ). Epistasis with daf-3 at 27° is complicated by the Hid phenotype of daf-3 mutants so we concentrated on epistasis with daf-5. Double mutants of unc-3, unc-31, and unc-64 with daf-3 were not Syn-Daf and exhibited the same epistasis relationships as the double mutants with daf-5 under conditions that permitted scoring of suppression (data not shown).

As shown in Table 10, mutations in daf-5 did not suppress the Hid phenotype of unc-64 or unc-31, suggesting that these genes act in parallel to the group II pathway. Mutations in daf-5 partially suppressed unc-3 or daf-7 at 26.6° but showed little suppression at a higher temperature. The lack of suppression seen at the highest temperatures may be due to inability to detect partial suppression when dauer formation is maximally induced. The similarity of unc-3 and daf-7 suppression by daf-5 suggests that unc-3 and daf-7 act at a similar position in the group II branch of the dauer pathway. The fact that daf-5 only partially suppresses the Daf-c phenotype of daf-7 at 27° while it completely suppresses the Daf-c phenotype at 25° suggests that there are outputs of the group II pathway at 27° that either do not exist at 25° or are not detectable. daf-5 mutations also only partially suppress daf-1 and daf-14 mutants at 27° (data not shown), consistent with the daf-7 results. Mutations in daf-5 showed no suppression of the group I Daf-c gene daf-11 at 27°.

The opposing phenotypes of daf-3 and daf-5 at 27° permitted us to perform epistasis on these two genes for the first time. We built double mutants of three different daf-3 alleles with mutations in daf-5. As shown in Table 10, mutations in daf-5 did not suppress the Daf-c phenotype of any of the daf-3 mutants, suggesting that daf-3 acts downstream of daf-5 in the group II pathway. This is consistent with the fact that daf-3 encodes a SMAD protein that may act in the nucleus as a transcription factor to directly regulate genes involved in dauer development (PATTERSON et al. 1997 ; THATCHER et al. 1999 ). Similar results were seen with the sa205 allele of daf-5 (data not shown). Finally, we observed partial suppression by daf-5 of the Hid phenotype of the Dyf mutant osm-6, consistent with osm-6 functioning in parallel to the group II branch of the dauer pathway.

Double mutants with daf-16: Mutations in daf-16 completely suppress the Daf-c phenotype at 25° of Daf-c mutants in the insulin branch of the dauer pathway (VOWELS and THOMAS 1992 ; GOTTLIEB and RUVKUN 1994 ; LARSEN et al. 1995 ; PARADIS et al. 1999 ). Mutations in daf-16 partially suppress group I Daf-c mutants and only very weakly suppress group II Daf-c mutants at 25° (VOWELS and THOMAS 1992 ). As shown in Table 11, mutations in daf-16 completely suppressed the Hid phenotype of unc-64 and unc-31 but only partially suppressed the Hid phenotype of unc-3. This suggests that unc-64 and unc-31 function in the insulin branch of the dauer pathway, while unc-3 probably functions in parallel, consistent with the daf-5 epistasis results. Mutations in daf-2 were only partially suppressed by mutations in daf-16 at 27°, suggesting that there are daf-16-independent outputs of the insulin signaling pathway at 27° that either do not exist at 25° or are not detectable. Mutations in daf-16 partially suppressed the Hid phenotype of daf-3 and three different Dyf mutants, osm-6, daf-10, and osm-5, consistent with these genes functioning in parallel to the insulin branch of the dauer pathway.

Double mutants with pdk-1(gf) and akt-1(gf): The pdk-1 and akt-1 genes function downstream of daf-2 and age-1 in the insulin branch of the dauer pathway, but upstream of daf-16 (Fig 1). Dominant gain-of-function mutations in either pdk-1 or akt-1 suppress the Daf-c phenotype of age-1 mutants at 25° but do not suppress daf-2 (PARADIS and RUVKUN 1998 ; PARADIS et al. 1999 ), suggesting that there is a bifurcation of the insulin signaling pathway downstream of daf-2. Since unc-64 and unc-31 appear to act in the insulin pathway, we built double mutants of unc-64(e246) and unc-31(e928) with the pdk-1(mg142) and akt-1(mg144) gain-of-function mutations. unc-64 and unc-31 double mutants with either pdk-1(mg142) or akt-1(mg144) formed 100% dauers at 27°, suggesting that unc-64 and unc-31 act upstream of the bifurcation in the pathway, downstream of pdk-1 and akt-1, or in the branch that does not consist of age-1, pdk-1, and akt-1. Alternatively, the gain-of-function mutations may not activate the pathway enough to suppress upstream Daf-c mutations at 27°.

Epistasis based on pheromone response at 25°:
As another method of positioning unc-64, unc-31, and unc-3 in the dauer pathway, we examined whether daf-5 could suppress dauer formation induced by a high level of pheromone at 25° in these mutants. As shown in Table 12, mutations in daf-5 completely suppressed the pheromone response of unc-3 and daf-7 but did not suppress the pheromone response of either unc-64 or unc-31. Similar results were seen with daf-3 in place of daf-5 (data not shown). This provides further evidence that unc-3 acts in the group II pathway and that unc-64 and unc-31 act in parallel. A daf-5; daf-11 double mutant also did not respond to pheromone. Since unc-64 and unc-31 double mutants with daf-5 responded normally to pheromone, this suggests that unc-64 and unc-31 do not act in the group I pathway.

One possible explanation for the failure to see suppression of the unc-64 or unc-31 pheromone responses by daf-5 is that dauer formation was so strongly induced by the high level of pheromone in this experiment that partial suppression could not be detected. To investigate this possibility, we assayed the daf-5; unc-64 and daf-5; unc-31 double mutants at a range of pheromone concentrations. As shown in Fig 4, at pheromone concentrations that induced an intermediate level of dauer formation, the daf-5; unc-64 and daf-5; unc-31 double mutants responded almost identically to the unc-64 and unc-31 single mutants. Thus, the lack of unc-64 and unc-31 suppression by daf-5 cannot be accounted for by mere quantitative differences between these genes and unc-3.

View larger version (18K): In this window In a new window Download PPT slide   Figure 4. Dauer formation of daf-5; unc-64 and daf-5; unc-31 double mutants in response to exogenous pheromone at 25°. Approximately 50–100 animals were counted at each concentration of pheromone. Both graphs use data from the same experiment but are plotted separately to facilitate comparison of the unc-64 and unc-31 single mutants with the daf-5 double mutants.

As a final method of assessing epistatic interactions, we assayed the Daf-d phenotype of double mutants of unc-64, unc-31, and unc-3 with either daf-3 or daf-5. The daf-3(e1376) and daf-5(e1385) mutants have a strong Daf-d phenotype at 20°, including a failure to form dauers in response to starvation. unc-64, unc-31, and unc-3 mutants form dauers readily when starved, at levels comparable to or greater than wild-type N2. Mutations in daf-3 and daf-5 completely abolished starvation-induced dauer formation of daf-7 or unc-3 mutants but had no discernible effect on starvation-induced dauer formation of unc-64 or unc-31 mutants (data not shown). This provides further evidence that unc-3 acts in the group II pathway and that unc-64 and unc-31 act in parallel.

Double mutants of unc-64, unc-31, and unc-3 with other Daf-c genes:
Double mutants of Daf-c genes in different branches of the dauer pathway have a stronger Daf-c phenotype than either single mutant, while double mutants of Daf-c genes in the same branch do not have an enhanced Daf-c phenotype (THOMAS et al. 1993 ; OGG et al. 1997 ). As a complementary approach to epistasis with Daf-d mutants, we built double mutants of unc-64, unc-31, and unc-3 with Daf-c mutants in each branch of the dauer pathway. Many of these double mutants formed 100% nonrecovering dauers at all temperatures, preventing the establishment of a strain. The incompletely penetrant Daf-c phenotype of daf-7 at 15° was enhanced to 100% by mutation of unc-3, unc-31, or unc-64, suggesting that these genes act in parallel to daf-7. This was expected for unc-64 and unc-31, which appear to act in the insulin branch of the pathway, but was unexpected for unc-3, which appeared to act in the group II pathway on the basis of the epistasis results presented above. Double mutants of unc-3 with the other group II Daf-c genes daf-1 and daf-14 also exhibited 100% dauer formation at 15°. Thus, although unc-3 may function upstream of daf-5 in the group II pathway, it must act at least partially in parallel to the group II Daf-c genes. unc-31 and unc-64 did not enhance the Daf-c phenotype of a daf-2 mutant at 15° (AILION et al. 1999 ), supporting the idea that these genes function in the insulin branch of the pathway.

Dominance of Daf-c genes at 27°:
Daf-c mutants with a strong Daf-c phenotype at 25° are recessive at this temperature, with the exception of the semidominant mutant daf-28 (MALONE and THOMAS 1994 ). daf-28 does not appear to act in any of the three branches of the dauer pathway depicted in Fig 1 (MALONE et al. 1996 ). We tested whether any of the 25° Daf-c mutants were dominant at the more strongly dauer-inducing temperature of 27°. As shown in Table 13, the daf-7(e1372) mutant was moderately dominant at 27°, while no other Daf-c mutants tested exhibited any dominance. The unc-64, unc-31, and unc-3 mutants also did not exhibit any dominance at 27° (AILION et al. 1999 and data not shown). To verify that daf-7 dominance was not allele specific, we tested the daf-7(m62) mutant and found that it also was partially dominant at 27° (data not shown). daf-7/+ heterozygotes exhibited a Daf-c phenotype at 27° regardless of whether the daf-7 mutant gene came from the male or hermaphrodite parent, indicating that this phenotype could not be accounted for by a maternal effect of daf-7. daf-7 encodes a TGF-ß-like protein that acts as a secreted ligand (REN et al. 1996 ). e1372 is a missense mutation and m62 is a nonsense mutation (REN et al. 1996 ), indicating that these are loss-of-function mutations and that dominance is caused by haploinsufficiency. Reducing the gene dosage of daf-7(+) would be expected to decrease the concentration of DAF-7 ligand. We hypothesize that at 27°, this decrease is below the threshold needed for nondauer signaling.

Expression of daf-7::gfp at 27°:
One possible explanation for the partial dominance of daf-7 at 27° is the fact that daf-7 expression is reduced by increased temperature (SCHACKWITZ et al. 1996 ). Perhaps, downregulation of daf-7 at 27° is significantly greater than at 25°, resulting in a Daf-c phenotype when daf-7 gene dosage is reduced. Previously, SCHACKWITZ et al. 1996 examined expression of the integrated daf-7::gfp array saIs7 and found that GFP was undetectable at 25°. To examine daf-7::gfp expression at 27°, we made use of the integrated array saIs8, which expresses GFP at considerably higher levels than saIs7. As shown in Table 14, the percentage of ASI neurons expressing daf-7::gfp remains roughly the same from 15° to 27°, but the percentage strongly expressing GFP drops considerably, particularly at 27°. This is consistent with the idea that the dominance of daf-7 mutants at 27° results from the greater reduction in daf-7 expression. Some differences in daf-7::gfp expression were seen between the left and right ASI neurons, but it is not clear whether these differences are significant. At temperatures >15°, there was a significant percentage of animals that expressed daf-7::gfp in cells other than ASI. The possible significance of this is also unclear.

Cell kills:
The identification of particular neurons involved in regulating dauer formation has been accomplished by killing identified neurons with a laser microbeam (BARGMANN and HORVITZ 1991 ; SCHACKWITZ et al. 1996 ). From these studies, the ASI and ADF neurons were shown to function as redundant dauer-repressing neurons. Killing both these neurons results in a Daf-c phenotype at 20°, but killing either ASI or ADF alone in a wild-type background does not lead to a Daf-c phenotype (BARGMANN and HORVITZ 1991 ). Killing ASI (but not ADF) alone in an unc-31 mutant results in a Daf-c phenotype (AVERY et al. 1993 ). To determine the involvement of ASI in dauer formation at 27°, we killed this cell in the wild-type N2 and assayed dauer formation at 27°. As shown in Table 15, killing ASI alone was sufficient to result in a Daf-c phenotype at 27°. This is similar to the genetics of the Syn-Daf phenotype where apparent redundancies at lower temperatures evaporate at 27°. We confirmed that killing ASI alone in an unc-31 mutant at 25° was sufficient to cause dauer formation and also showed that killing ASI alone was sufficient to cause dauer formation at 25° in an unc-64 mutant.

unc-3 encodes a transcription factor expressed only in ASI and ventral cord motor neurons (PRASAD et al. 1998 ). Thus, the dauer phenotype of unc-3 mutants is expected to have a site of action in ASI and it was possible that the unc-3 mutation leads to misfunction of ASI equivalent to an ASI cell kill. Evidence in support of this idea is that (1) unc-3 is Daf-c at 27° and killing ASI leads to a Daf-c phenotype at 27°; (2) unc-31 and unc-64 are Syn-Daf with unc-3 or with an ASI cell kill; and (3) unc-3 appears to function with the group II pathway, which is thought to act through ASI (SCHACKWITZ et al. 1996 ). Killing ASI did not cause dauer formation in an unc-3 mutant (Table 15), consistent with the idea that unc-3 perturbs ASI function. However, killing ADF did not cause dauer formation in the unc-3 mutant either, as would be expected if the unc-3 mutant was equivalent to an ASI cell kill. Since ADF was killed in only three animals, this result should be interpreted cautiously. Thus, while it is probable that ASI does not function properly in an unc-3 mutant, it likely retains some of its ability to regulate dauer formation.

Male dauer formation:
In the course of performing crosses with unc-3 mutants at 20°, we observed dauers after mating wild-type males to unc-3 hermaphrodites. Since unc-3 maps to the X chromosome, we hypothesized that these might be unc-3 male dauers. To test this idea, we picked these dauers and allowed them to recover to score their sex. All such dauers were male, confirming our hypothesis. At 20°, 38% of the unc-3(e151) males formed dauers and 0% of the unc-3 hermaphrodites formed dauers. Thus, there is differential regulation of dauer formation in unc-3 males and hermaphrodites.

To investigate whether the increased dauer formation of males was specific to unc-3, we assayed dauer formation of N2 wild-type males and hermaphrodites in response to pheromone at 25°. As shown in Fig 5, males showed much stronger dauer formation in response to pheromone than hermaphrodites. Thus, males appear to be generally more sensitized to dauer-inducing conditions. The increased frequency of male dauer formation in several Daf-c mutants has been noted previously (VOWELS and THOMAS 1992 ). Males carry one X chromosome while hermaphrodites carry two X chromosomes. To determine if the number of X chromosomes was responsible for the dauer formation differences observed between males and hermaphrodites, we examined tra-2(q276) males, which are phenotypically male, but carry two X chromosomes. tra-2(q276) XX males exhibited a hypersensitive response to pheromone similar to N2 XO males (data not shown), indicating that the increased male response comes as a result of being phenotypically male and not from the number of X chromosomes. We examined male dauer formation of unc-64 and unc-31 in double mutants with him-5. Unlike unc-3, unc-31 and unc-64 males did not form dauers at 20°. Since the unc-64 and unc-31 hermaphrodite Daf-c phenotype appears to be at least as strong as that of unc-3, this difference is unlikely to be merely quantitative. The increased sensitivity of males to dauer-inducing conditions suggests that males have either additional dauer-inducing pathways not present in hermaphrodites or an enhanced response by the same pathways.

View larger version (18K): In this window In a new window Download PPT slide   Figure 5. Males are hypersensitive to pheromone. The graph plots dauer formation of wild-type males and hermaphrodites in response to exogenous pheromone at 25°. The progeny of mated N2 hermaphrodites were grown on different concentrations of pheromone. The number of dauers and male and hermaphrodite nondauers was counted. Dauers were then picked to plates without pheromone at 15°. After dauers recovered, they were scored as either male or hermaphrodite. Approximately 50–100 animals of each sex were counted at each concentration of pheromone.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Dauer formation is strongly induced at 27°:
Previous work showed that dauer formation is induced more at 25° than at 15° (GOLDEN and RIDDLE 1984A , GOLDEN and RIDDLE 1984B ). Here we show that 27° is a much more strongly inducing dauer stimulus than 25°. Wild-type worms show a strong- er increase in dauer induction by pheromone over the 2° temperature change from 25° to 27° than they do over the 10° temperature change from 15° to 25°. Thus, induction of dauer formation at 27° is likely a specific nonlinear response to this temperature and not due to general thermodynamic effects on biological processes, which should increase linearly with increasing temperature (in degrees Kelvin). Since wild-type worms are highly sensitive to pheromone at 27°, the thermal input to dauer formation must be acting at least partially in parallel to the pheromone response pathways. The thermal input to dauer formation at 27° could either be a more extreme version of the thermal input received at lower temperatures or it could be a distinct input. Over the temperature range from 15° to 25°, dauer phenotypes exhibit quantitative differences, with dauer formation becoming stronger as the temperature is increased. Since dauer phenotypes at 27° are not merely quantitatively stronger than phenotypes at 25° but show qualitative differences (see below), we favor the hypothesis that 27° provides a dauer-inducing input partially distinct from the input received at 25° or lower temperatures. Thus, there may be sensory pathways and cells dedicated to detecting a 27° stimulus. This of course does not preclude that a 27° stimulus would also affect pathways that receive temperature input at lower temperatures. Many organisms have distinct thermoreceptors that are activated at different temperatures, rather than a single thermoreceptor whose activity is modulated over a wide temperature range (SPRAY 1986 ). Thus, perception of any given temperature occurs by a combinatorial mechanism of activating multiple thermoreceptors. In vertebrates, sensation of noxiously high temperatures is mediated by the capsaicin receptor (CATERINA et al. 1997 ). osm-9 encodes the closest C. elegans homolog of the capsaicin receptor (COLBERT et al. 1997 ). The osm-9(n1601) null mutant had the same phenotype as N2 at 27° (data not shown), suggesting that sensation of the 27° stimulus occurs by a different mechanism.

A cellular pathway that responds to temperature in C. elegans has been defined for thermotaxis behavior (MORI and OHSHIMA 1995 ). In this pathway, AFD appears to be a primary thermosensory cell. Mutations in the ttx-1 gene cause defects in the structure of AFD (specific to the proposed thermosensory structures) and severe defects in thermotaxis behavior. We show here that ttx-1 mutants induce dauer formation normally in response to temperature over the entire range from 15° to 27°, but are hypersensitive to dauer pheromone at all temperatures. Mutations in the ttx-3 gene affect the function of AIY, an interneuron in the thermotaxis pathway (MORI and OHSHIMA 1995 ). ttx-3 mutations cause severe defects in thermotaxis but affect dauer formation only mildly; additionally, the dauer effect appears to be specific to recovery, not formation of dauers (HOBERT et al. 1997 ). Thus, thermal inputs that regulate dauer formation appear to be at least in part distinct from thermal inputs that regulate thermotaxis. C. elegans also exhibits a thermal avoidance behavior at even higher temperatures (WITTENBURG and BAUMEISTER 1999 ). Like dauer formation, thermal avoidance behavior appears to have thermal inputs distinct from the thermotaxis circuit. Thus, C. elegans appears to resemble other organisms in having multiple thermoreceptors that respond to different temperatures.

In addition to strongly inducing dauer formation in wild-type animals, growth at 27° leads to a strongly penetrant Daf-c phenotype in several mutants (unc-64, unc-31, and unc-3) that do not exhibit any Daf-c phenotype on their own at 25°. Double mutants of these genes exhibit a Syn-Daf phenotype at 25°. Such a synthetic phenotype could have indicated full genetic redundancy, but in this case the synthetic phenotype appears to result from a combination of weak phenotypes that are not detectable at 25°. These mutants would have been difficult to isolate in screens for Daf-c mutants at 25° since they would require the simultaneous occurrence of two mutations. The finding that these mutants have strong single mutant phenotypes at 27° allows for an efficient way to identify new dauer genes that could not have been isolated in screens performed at 25°. We have performed such a screen for Daf-c mutants at 27°, and we isolated several new alleles of unc-31 and unc-3 as well as a number of new dauer genes (M. AILION and J. H. THOMAS, unpublished results).

High temperature is sufficient to induce dauer formation:
At temperatures of 25° or lower, pheromone is both necessary and sufficient to induce dauer formation. At 27°, wild-type and pheromone-deficient daf-22 animals are capable of forming dauers in the absence of exogenous pheromone when food is plentiful, a phenotype not seen at lower temperatures. This implies that the more extreme temperature is a sufficient stimulus to induce dauer formation, unless there is a novel pheromone production pathway operative at 27° that does not depend on daf-22 gene activity. The fact that Dyf mutants, which block pheromone detection, are Daf-c at 27° is consistent with the possibility that dauer formation at 27° is pheromone independent.

Pheromone is likely to act as a measure of population density. Since high temperatures appear sufficient to induce dauer formation on their own, worms in the wild probably encounter hot temperatures at low population densities, where dauer formation is dictated by the stressful thermal stimulus rather than a lack of resources, as occurs with overcrowding. We note that N2 wild-type animals form dauers only transiently at 27°. Why induce dauer formation if only to recover immediately? There are several possible explanations that are not mutually exclusive. One possibility is that pheromone concentrations are artificially low in the lab growth conditions of high food concentrations and relatively few animals on a naive plate that had no time to accumulate endogenously produced pheromone by earlier generations of animals. Since very low concentrations of pheromone are effective at inducing nontransient dauer formation at 27°, it is easy to imagine that such low levels of pheromone might usually be present in natural environments. A second possibility similar to the first is that recovery of dauers at 27° is especially sensitive to decreased amounts of food. The concentration of food present in our laboratory assays is probably rarely achieved in nature. Perhaps with reduced amounts of food more likely to mimic natural conditions, high temperature may be sufficient to induce dauer formation and inhibit recovery. A third possibility is that temperatures >27° may be sufficient to induce dauer formation and inhibit recovery. A fourth possibility is that dauer formation really is more sensitive to temperature than dauer recovery and that this has biological significance. Inducing dauer formation transiently may be advantageous in highly variable environments that change rapidly. By inducing dauer formation at temperatures that are dangerous but not lethal, the animals may be "hedging their bets" against future expectations. If conditions continue to worsen, animals can inhibit recovery once they have achieved the dauer stage, but since the decision to form a dauer must be made beginning at the L1 molt (SWANSON and RIDDLE 1981 ; GOLDEN and RIDDLE 1984A ), it would be too late to induce dauer formation if they had not already done so. On the other hand, if conditions stay the same or improve, animals that only transiently induced dauer formation will have a "head start" over animals that arrested at the dauer stage. Such a head start would lead to earlier reproduction and could be essential for progeny survival in a highly competitive resource-limited environment. While many genes that regulate dauer formation also regulate dauer recovery, there is evidence for some differential regulation of the two processes (MALONE et al. 1996 ; HOBERT et al. 1997 ; SZE et al. 2000 ; TISSENBAUM et al. 2000 ).

Genes with positive and negative influences on dauer formation:
One of the unexpected findings of this study was that many genes have opposing positive and negative influences on regulation of dauer formation, as revealed by their 27° phenotypes. Mutations in any of the large group of Dyf genes, which affect the structure of the ciliated endings of sensory neurons, result in a Daf-d phenotype and nonresponsiveness to pheromone at 25°, but a strong Daf-c phenotype at 27°. This reversal of a Daf-d phenotype is also seen for mutants in the daf-3 gene, which occupies a distinct position in the pathway. Here we discuss several different possibilities that could account for such reversals of phenotype and suggest that different mechanisms may be operating in the different cases observed.

How do genes that are Daf-d at 25° become Daf-c with only a 2° increase in temperature? For the Dyf genes, we favor the following hypothesis. At temperatures of 25° or lower, pheromone is necessary to induce dauer formation. The Dyf mutants have defects in the structure of the amphid neuron endings exposed to the environment where pheromone detection occurs. These structural defects prevent pheromone detection and hence lead to a Daf-d phenotype. However, at 27°, detection of pheromone is no longer necessary to induce dauer formation. Perhaps the basal activity state of the amphid neurons is different in the Dyf mutants. This altered basal activity may be insufficient to induce dauer formation at 25°, but it may be above the threshold for constitutive dauer formation at 27°. Support for this idea comes from analysis of mutants with amphid structural defects, but which affect cells other than the neurons themselves. For example, the daf-6 mutant has defects in the amphid sheath cell that lead to a Dyf phenotype and the inability to respond to dauer pheromone (ALBERT et al. 1981 ; HERMAN 1984 , HERMAN 1987 ; PERKINS et al. 1986 ). daf-6 mutants are not Daf-c at 27°, suggesting that the inability to sense the environment per se does not lead to an altered basal neuronal activity. Since Dyf mutants remain pheromone insensitive at 27°, it is unlikely that there are additional pheromone-sensing pathways at 27° that do not operate at 25°. Since there are both dauer-repressing and dauer-promoting neurons, the Daf phenotype of Dyf mutants at any given temperature could depend on a balance of the activities of different cells. This model could also help explain the Daf-c phenotype of daf-19 mutants. daf-19 mutants completely lack sensory cilia, but unlike all other Dyf mutants, daf-19 mutants are Daf-c at all temperatures (PERKINS et al. 1986 ; SWOBODA et al. 2000 ). Perhaps daf-19 mutations more severely alter the basal activity of the amphid neurons so that it is above the threshold to induce dauer formation independently of the pheromone input at all temperatures.

Does a similar explanation account for the reversal of the daf-3 mutant phenotype? Mutations in daf-3 could have both positive and negative influences on the activity of some neurons, perhaps altering the basal activity but preventing inducibility by pheromone. However, we think that this is unlikely in the case of daf-3 for two reasons. First, daf-3 mutants are Daf-c at 27° while daf-5 mutants are not. If the 27° Daf-c phenotype of daf-3 were a nonspecific characteristic of mutants in this part of the pathway (as is the case for the Dyf mutants), we would expect daf-3 and daf-5 to behave identically at 27°, as they behave identically in regulating dauer formation at lower temperatures. Second, daf-3 and daf-5 mutants have normal pheromone sensitivity at 27°. Thus, pheromone response pathways that act in parallel to daf-3 and daf-5 must be sufficient for dauer formation at 27°. The different 27° phenotypes of daf-3 and daf-5 suggest that there may be specific regulation of daf-3 activity at 27°. daf-5 has not yet been cloned, but the molecular identification of the DAF-3 gene product as a SMAD transcription factor (PATTERSON et al. 1997 ) suggests a few specific possibilities. DAF-3 is hypothesized to act in the nucleus as a transcription factor either to activate genes involved in dauer development or to repress genes involved in nondauer development. Its ability to translocate to the nucleus and bind DNA may depend on positive or negative regulation by partner SMAD proteins or other transcription factors (ATTISANO and WRANA 2000 ). One of several ways to explain the reversal of the daf-3 mutant phenotype posits that DAF-3 binds to the same target genes at 25° and 27° but forms different partnerships that make it an activator of transcription at one temperature and a repressor at the other. For example, mutations in daf-3 could lead to excessive transcription of dauer-promoting genes at 27° and reduced transcription of the same target genes at 25°.

The unc-31 gene was also shown to have both positive and negative effects on dauer formation. Unlike the Dyf and daf-3 mutants, unc-31 mutants are not Daf-d at any temperature and the reversal of phenotype is seen at lower temperatures. unc-31 mutants have a reduced sensitivity to dauer pheromone at 22° and an increased sensitivity at 25°. unc-31 clearly has both dauer-promoting and dauer-repressing effects simultaneously, since at 22° it exhibits a reduced response to dauer pheromone but also strongly enhances the Daf-c phenotype of other Syn-Daf genes. The simplest explanation for these effects is that unc-31 functions in different cells to promote or inhibit dauer formation, and that the balance of opposing inputs can be tilted in either direction depending on environmental stimuli or mutation of other genes in parallel pathways. This hypothesis is similar to the hypothesis presented above for the Dyf genes, except in that case the opposing forces were proposed to act on the activity of the same cell, but in different ways. Support for the idea that unc-31 functions in multiple cells comes from the observation that an unc-31::lacZ reporter is expressed throughout the nervous system (LIVINGSTONE 1991 ). Furthermore, unc-31 encodes a homolog of Ca²⁺-dependent activator protein for secretion (CAPS), a protein that regulates exocytosis of dense-core vesicles and likely affects signaling throughout the nervous system (LIVINGSTONE 1991 ; WALENT et al. 1992 ; ANN et al. 1997 ).

The tax-4 and tax-2 genes, which encode subunits of a CNG ion channel (COBURN and BARGMANN 1996 ; KOMATSU et al. 1996 ), have both dauer-promoting and dauer-repressing effects that have been noted previously (COBURN et al. 1998 ). Interpretation of the tax-4/tax-2 results is complicated by the fact that several of the alleles are clearly not null and may have gain-of-function phenotypes. Additionally, it is not clear what happens in vivo to channel activity in the absence of one subunit, especially given the presence of other potential channel subunits encoded by the genome (BARGMANN 1998 ). Nevertheless, from the previous data and the results presented here, we can draw several striking conclusions.

At 25°, tax-4 and tax-2 mutations suppress the dauer phenotype of group I Daf-c mutants and enhance the dauer phenotype of group II Daf-c mutants (COBURN et al. 1998 ). Thus, tax-4 and tax-2 have properties of both group I Daf-d and Daf-c genes. The tax-4(ks11) mutant has a Daf-c phenotype on its own at 25° and resembles the group I Daf-c genes in its genetic interactions at this temperature. This suggests that it may act in ASJ as a target of cGMP made by the DAF-11 guanylyl cyclase. However, ASJ is a dauer-promoting neuron, and the loss of a nonspecific cation channel such as tax-4 would be expected to hyperpolarize the neuron, thereby inhibiting it and reducing dauer formation, the opposite of the phenotype observed for the tax-4(ks11) mutant. This highlights the problem of explaining the daf-11 mutant phenotype in ASJ as the result of effects on a cyclic nucleotide-gated channel. The daf-11 mutant should have reduced levels of cGMP, which in any scenario with a wild-type CNG channel as the target would lead to hyperpolarization of the cell. In a dauer-promoting cell, this would lead to reduced dauer formation, the opposite of the daf-11 Daf-c phenotype, unless the dauer-promoting cell is an unusual neuron that transmits signals when hyperpolarized. Clearly, something is missing from the picture. One obvious possibility is that daf-11 functions in dauer-repressing neurons in addition to ASJ.

At 27°, all tax-4 mutants appear to be strongly Daf-c. Of tax-2 mutants, only p691 has an equally strong Daf-c phenotype. Interestingly, this allele mutates the identical proline residue in the channel pore mutated in the strongest Daf-c allele of tax-4, ks11. The tax-2(p694) mutation eliminates expression of the TAX-2 subunit from four neurons (AFD, ASE, ADE, and BAG) but has normal expression and function in the other seven neurons that express the channel (COBURN and BARGMANN 1996 ). Since this mutant is not Daf-c at 27°, it suggests that the tax-2(p691) 27° Daf-c phenotype has a site of action in one or more of the seven expressing neurons (which include ASJ and ASI). However, the tax-2(p694) mutation suppresses the 25° Daf-c phenotypes of tax-4(ks11) and daf-11 (COBURN et al. 1998 ), suggesting that the group I Daf-d phenotype has a site of action in one of the cells AFD, ASE, ADE, or BAG, none of which have been previously implicated in regulating dauer formation. Thus, there is evidence that the Daf-c and Daf-d phenotypes of tax-2 have different cellular sites of action. tax-2(p694) does not suppress tax-4 Daf-c phenotypes at 27°, suggesting that there may be different cellular sites of action for tax-4 Daf-c phenotypes at 25° and 27° or different dependence on TAX-2 subunits.

Since tax-4 encodes an -subunit that can form homomeric ion channels in the absence of ß-subunits (KOMATSU et al. 1996 , KOMATSU et al. 1999 ), tax-2 phenotypes are expected to be weaker than tax-4 phenotypes. For the Daf-c phenotype, tax-4 mutants clearly appear to be stronger than tax-2 mutants as expected. However, tax-2 mutants show an unexpectedly stronger defect in response to dauer pheromone. This defect is seen in all tax-2 alleles, including p694, suggesting that one or more of the cells AFD, ASE, ADE, or BAG may be involved in pheromone sensation. Since tax-2 mutants show a stronger defect than tax-4 mutants, tax-2 may function in the absence of tax-4. Other evidence to suggest that TAX-2 has functions in the absence of TAX-4 is that tax-2 mutations can suppress either the dauer formation or recovery defects of tax-4 mutants, including the putative tax-4 null allele p678. If tax-2 mutations only eliminated the function of the TAX-4/TAX-2 channel, there should be no effect of tax-2 mutations in a tax-4 null background. This suggests that TAX-2 has functions independently of TAX-4, either by itself or in partnership with other CNG channel subunits. C. elegans appears to have four other CNG channel subunits encoded in its genome, all of which appear to be -subunits, supporting this idea (BARGMANN 1998 ; data not shown).

Implications for the dauer genetic pathway:
We placed unc-64, unc-31, and unc-3 in the dauer pathway by performing epistasis with Daf-d genes under several different conditions. The 27° Daf-c phenotype of unc-64 and unc-31 mutants was completely suppressed by mutations in daf-16 but neither the Daf-c nor pheromone response of these mutants was suppressed at all by mutations in daf-5. These data support the conclusion that unc-64 and unc-31 act in the insulin branch of the dauer pathway as suggested previously (AILION et al. 1999 ). In addition to regulating dauer formation, the insulin branch regulates adult longevity (KENYON et al. 1993 ; DORMAN et al. 1995 ; LARSEN et al. 1995 ). Consistent with a function in the insulin branch, unc-64 and unc-31 mutants have extended life spans (AILION et al. 1999 ). unc-64 and unc-31 encode homologs of syntaxin and CAPS, proteins that mediate Ca²⁺-regulated secretion (LIVINGSTONE 1991 ; ANN et al. 1997 ; OGAWA et al. 1998 ; SAIFEE et al. 1998 ), and they have been proposed to function in the regulation of insulin secretion (AILION et al. 1999 ). unc-64 and unc-31 are not suppressed by gain-of-function mutations in pdk-1 or akt-1, which function in one branch of a divergent pathway downstream of the DAF-2 insulin receptor. This is consistent with unc-64 and unc-31 functioning upstream of daf-2 or downstream of daf-2 in the other branch.

Unlike unc-64 and unc-31, the 27° Daf-c phenotype of unc-3 is partially suppressed by mutations in daf-5 while the pheromone response at 25° is completely suppressed. This suggests that unc-3 acts in the group II Daf-c pathway that consists of a TGF-ß signaling cascade. Consistent with this, unc-3 encodes a transcription factor expressed in the sensory neuron ASI (PRASAD et al. 1998 ). Killing ASI is not sufficient to cause a Daf-c phenotype in unc-3 mutants at 25°, while it is sufficient to cause a Daf-c phenotype in unc-64 and unc-31 mutants at 25°, providing further evidence that unc-3 functions in ASI but that unc-64 and unc-31 act in a parallel pathway. unc-3 probably also acts partly in parallel to the group II pathway since unc-3 mutations enhance the Daf-c phenotype of group II mutants at 15°.

unc-3 mutants are partially suppressed by daf-16 at 27°. daf-16 mutations also partially suppress the 27° Daf-c phenotype of daf-3 and Dyf mutants. Thus, partial suppression by daf-16 at 27° appears to be a nonspecific phenomenon that probably results from effects on a parallel pathway that converges further downstream. This is similar to the partial suppression of group I Daf-c mutants by daf-3 and daf-5 at 25°. The partial suppression of Dyf, daf-3, and unc-3 mutants by daf-16 at 27° contrasts with the complete suppression of unc-64 and unc-31. Since unc-64 and unc-31 mutants have stronger Daf-c phenotypes at 27° than unc-3, daf-3, and Dyf mutants, the partial suppression by daf-16 of these latter mutants cannot be explained by mere quantitative differences. It was reported by others that daf-16 mutations completely suppress the Daf-c phenotype of Dyf mutants at 27° (APFELD and KENYON 1999 ). Several possibilities could account for the discrepancy between their results and ours. First, they use a different allele of daf-16 in their experiment that could give stronger suppression. While we have not performed epistasis with the daf-16 null allele they used, we have performed epistasis of unc-64 and unc-31 with both m27 (the allele used in our Dyf epistasis experiments) and mgDf50, a different null allele, and both behave identically. Thus, while we have not ruled out the possibility that m27 is a weaker allele for suppression of Dyf mutants, it behaves like a null allele in suppression of the stronger Daf-c phenotypes of unc-64 and unc-31. Two more likely possibilities for the discrepancy are that their assays were performed at a slightly lower temperature at which partial suppression appears complete or that they failed to detect daf-16 partial dauers, which recover more rapidly. Our results do not support the model proposed by APFELD and KENYON 1999 that the Dyf mutants block specific sensory input to the insulin branch of the pathway. It seems more likely that the Dyf mutants act largely in parallel to the insulin branch, though it is possible that there is a direct effect of Dyf mutations on the activity of the insulin branch in addition to their effects on parallel pathways.

Reexamining the epistatic interactions of strong Daf-c genes at 27° leads to several new findings. First, mutations in daf-16 completely suppress daf-2 dauer formation at 25°, but only partially suppress it at 27°. This suggests that daf-2 has daf-16-independent outputs at 27°. This additional branch of the pathway downstream of daf-2 may either not exist at 25° or may not be stimulated enough to be detected. Similarly, mutants in the group II Daf-c genes are completely suppressed by daf-5 mutations at 25° but only partially suppressed at 27°. This suggests that there may also be an additional branch of the group II Daf-c pathway detected at 27° that acts in parallel to daf-5. Suppression of group II Daf-c phenotypes by daf-5 mutations illustrates several interesting points on interpreting epistasis results. At 25°, daf-5 completely suppresses daf-7. At temperatures near 27°, daf-5 partially suppresses daf-7. At slightly higher temperatures, daf-5 shows almost no suppression of daf-7. Thus, depending on the temperature of the assay, daf-5 could be interpreted as completely suppressing or not suppressing at all. Is this simply a quantitative difference in suppression at different temperatures? Epistasis of dauer formation induced by pheromone at 25° suggests not. The wild-type strain N2 forms almost 100% dauers on high levels of pheromone at 25°, but forms <20% dauers at 27° without pheromone. Thus, in a wild-type background, pheromone at 25° is a stronger dauer-inducing stimulus than 27° alone. If daf-5 mutations failed to suppress group II Daf-c mutants at 27° because the dauer-inducing stimulus was too strong, one would predict that daf-5 mutations would be even less effective at suppressing group II Daf-c mutants on pheromone at 25°. This is not the case; mutations in daf-5 completely suppress dauer formation in group II mutants on pheromone at 25°. Thus, although pheromone is a stronger dauer-inducing stimulus for wild type, 27° is a stronger dauer-inducing stimulus for daf-5 mutants. The epistasis result does not correlate to intrinsic strength of the stimulus but shows qualitative differences depending on the environmental conditions of the assay and the genetic mutations present in the strains. Gene interactions inferred from epistasis experiments performed under one set of conditions may not be the same as those under a different set of environmental conditions. In complex regulatory pathways responding to multiple inputs, such differences are likely to be common.

	ACKNOWLEDGMENTS

We thank Garth Patterson, Suzanne Paradis, and Gary Ruvkun for providing the daf-3(mgDf90), daf-3(mg125), daf-3(mg132), akt-1(mg144), and pdk-1(mg142) mutants; Jennifer Vowels, Elizabeth Malone Link, Kouichi Iwasaki, and Carole Weaver for constructing some of the Syn-Daf double and triple mutant strains; Wendy Schackwitz for help with cell identifications; Takao Inoue and Cori Bargmann for helpful discussions; and Robert Choy, Josh McElwee, and Elizabeth Newton for comments on the manuscript. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health (NIH) National Center for Research Resources. M.A. was a Howard Hughes Medical Institute Predoctoral Fellow. This work was supported by NIH grant R01GM48700.

Manuscript received May 10, 2000; Accepted for publication June 19, 2000.

	LITERATURE CITED

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