Suppressors of Transforming Growth Factor-ß Pathway Mutants in the Caenorhabditis elegans Dauer Formation Pathway

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

Communicating editor: P. ANDERSON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

The dauer is a developmentally arrested alternative third larval stage of Caenorhabditis elegans. Entry into this state is regulated by environmental cues, including temperature, food, and the concentration of constitutively secreted dauer pheromone. Genetically, three parallel pathways have been found that regulate this process. Of these, the group 2 pathway, which includes the genes daf-1, daf-3, daf-4, daf-5, daf-7, daf-8, and daf-14, mediates the transduction of environmental signals through the ASI chemosensory neuron and encodes a TGF-ß-related signaling pathway. To identify additional genes that function in this pathway, we carried out a screen for suppressors of mutations in daf-1, daf-8, and daf-14. From the total of 36 mutations, seven complementation groups were identified. Three complementation groups correspond to the previously described genes daf-3, daf-5, and daf-12. Three correspond to novel genes scd-1, scd-2, and scd-3. Genetic analysis of these scd genes is presented here. A fourth complementation group was represented by a single mutation sa315, which affects the daf-2/age-1 insulin-related signaling pathway.

THE dauer is a developmentally arrested third stage larva of the nematode Caenorhabditis elegans (CASSADA and RUSSELL 1975 ). Entry into dauer arrest is promoted by lack of food, high temperature, and high concentration of dauer pheromone, a constitutively secreted substance serving as an indicator of population density (GOLDEN and RIDDLE 1982 , GOLDEN and RIDDLE 1984A , GOLDEN and RIDDLE 1984B ). In the L1 and L2 larval stages, these environmental cues are sensed in part by chemosensory neurons in the amphid sensory organs in the head (BARGMANN and HORVITZ 1991 ; SCHACKWITZ et al. 1996 ). These neurons in turn relay the environmental information to a complex regulatory system that makes the critical decision to proceed to the dauer or the L3 stage.

Genetic analysis of dauer formation in C. elegans has focused on two classes of mutations (RIDDLE et al. 1981 ). The first class, exhibiting the Daf-c (dauer formation constitutive) phenotype, forms dauers inappropriately under dauer-noninducing conditions. Most mutations of this class are temperature sensitive. This conditionality is a consequence of the inherent temperature sensitivity of the dauer-formation process and does not result from temperature-sensitive gene products (MALONE and THOMAS 1994 ). The second class, exhibiting the Daf-d (dauer formation defective) phenotype, fails to form dauers under dauer-inducing conditions. Double mutant analyses placed these genes into a genetic pathway with three parallel branches (THOMAS et al. 1993 ; GOTTLIEB and RUVKUN 1994 ). Although the cellular sites of action of these genetic pathways are not known in detail, they probably correspond to parallel streams of sensory information that feed into the decision to make a dauer.

The first branch, called the group 1 pathway, includes the Daf-c genes daf-11 and daf-21 (THOMAS et al. 1993 ; BIRNBY et al. 2000 ). daf-11 encodes a receptor guanylyl cyclase. daf-21 encodes an HSP-90 heat-shock protein and is defined by a Daf-c gain-of-function mutation. Laser ablation studies have suggested that the function of these genes is associated with the chemosensory neuron ASJ (SCHACKWITZ et al. 1996 ).

The second branch, called the group 2 pathway or the DAF-7/transforming growth factor (TGF)-ß pathway, includes the Daf-c genes daf-1, daf-4, daf-7, daf-8, and daf-14 and the Daf-d genes daf-3 and daf-5 (THOMAS et al. 1993 ). Together, these genes encode a TGF-ß-related signaling cascade. Specific components are DAF-7 (TGF-ß/BMP related ligand; REN et al. 1996 ), DAF-1 (type 1 receptor serine/threonine kinase; GEORGI et al. 1990 ), DAF-4 (type 2 receptor serine/threonine kinase; ESTEVEZ et al. 1993 ), DAF-8, DAF-14, and DAF-3 (Smads; PATTERSON et al. 1997 ; RIDDLE and ALBERT 1997 ; INOUE and THOMAS 2000 ). The molecular identity of the daf-5 gene product has not yet been determined. By analogy with vertebrate and Drosophila TGF-ß-related signaling (MASSAGUE 1998 ), ligand binding probably induces multimerization of receptors and the activation of the type 1 kinase through phosphorylation. Activated type 1 kinase in turn probably phosphorylates downstream Smad proteins, which transduce the signal to the nucleus and regulate transcription. In the group 2 pathway, the DAF-3 Smad has been demonstrated to exhibit a sequence-specific DNA-binding activity and probably functions as a downstream transcription factor (THATCHER et al. 1999 ).

Genetically, mutations in the Daf-d genes daf-3 and daf-5 suppress mutations in all five Daf-c genes (VOWELS and THOMAS 1992 ). Furthermore, four of five group 2 Daf-c genes, daf-1, daf-7, daf-8, and daf-14, display essentially identical pleiotropies, which include Egl (egg-laying defective), Din (dark intestine), and Cpy (clumpy; the tendency of animals to aggregate and form clumps on plates) phenotypes (TRENT et al. 1983 ; THOMAS et al. 1993 ). Although daf-3 and daf-5 mutants display no obvious pleiotropies, they suppress all pleiotropies in double mutant combinations with daf-1, daf-7, daf-8, or daf-14.

Laser ablation and expression studies indicate that this pathway directly mediates transduction of environmental signals, regulating dauer formation through the chemosensory neuron ASI (BARGMANN and HORVITZ 1991 ; SCHACKWITZ et al. 1996 ). That this pathway is at least partly in parallel to the group 1 pathway is supported by the genetic evidence that daf-3 and daf-5 mutants do not strongly suppress daf-11 and daf-21. Conversely, mutations that suppress the Daf-c phenotype of daf-11 and daf-21 do not suppress group 2 Daf-c mutants (THOMAS et al. 1993 ).

The third pathway, here referred to as the daf-2/age-1 pathway, includes daf-2 (encoding an insulin/IGF1 receptor homolog; KIMURA et al. 1997 ), daf-16 (forkhead transcription factor; LIN et al. 1997 ; OGG et al. 1997 ), daf-18 (PTEN phosphatase; OGG and RUVKUN 1998 ; GIL et al. 1999 ; MIHAYLOVA et al. 1999 ; ROUAULT et al. 1999 ), age-1 (PI3 kinase; MORRIS et al. 1996 ), akt-1 and akt-2 (two isoforms of AKT protein kinase; PARADIS and RUVKUN 1998 ), and pdk-1 (PDK protein kinase; PARADIS et al. 1999 ). Loss-of-function mutations in daf-2, age-1, and pdk-1 cause a Daf-c phenotype. RNAi (double stranded RNA interference) of akt-1 and akt-2 together also produces a Daf-c phenotype. Loss-of-function mutations in daf-16 and daf-18 are Daf-d and suppress mutations in age-1 and/or daf-2. Dominant gain-of-function alleles of akt-1 and pdk-1 also exist and suppress mutations in age-1. Genetic interactions among daf-2/age-1 pathway genes are complex and do not always follow the pattern expected from a linear pathway. One interesting feature of daf-2/ age-1 pathway mutations is their tendency to cause formation of partial dauers, animals that exhibit only some characteristics of a dauer. This type of animal has not been observed in the wild type or in group 1 and group 2 pathway mutants.

Finally, daf-12 encodes a nuclear hormone receptor that functions at a unique downstream position in the dauer pathway (YEH 1991 ). Loss-of-function daf-12 alleles are Daf-d and suppress Daf-c mutations in all three pathways, suggesting that this gene functions downstream of the convergence of the three parallel pathways.

Here, we describe the result of screens for suppressors of the group 2 Daf-c mutations daf-1(sa184), daf-8(sa234), and daf-14(m77). Three new genes were identified, each represented by multiple alleles. In addition, a single mutation affecting the daf-2/age-1 pathway was isolated and analyzed.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Manipulation of C. elegans and nomenclature:
Maintenance and manipulation of C. elegans were as described (BRENNER 1974 ). All strains described here are in the N2 Bristol strain background, with the exception of a scd-2(sa935) strain used for some complementation tests. For clarity, genotypes of all double mutant strains are listed in the order daf-c; sup (rather than in the order of chromosomal locations as suggested by the standard C. elegans nomenclature system; HORVITZ et al. 1979 ).

Isolation of suppressor mutations:
daf-14(m77) and daf-8-(sa234) were chosen as representative alleles because they were the strongest daf-14 and daf-8 alleles available at the time. daf-1(sa184) was chosen because of high penetrance and because this mutation does not exhibit strong maternal rescue like daf-1(m40) (MALONE et al. 1996 ). Molecular data and phenotype analysis suggest that daf-14(m77) and daf-8(sa234) alleles may be null (INOUE and THOMAS 2000 ); however, these alleles are less penetrant than daf-1(sa184) and presumably do not completely block DAF-7/TGF-ß signaling. At restrictive temperatures (25°), all of these alleles caused 99–100% dauer formation, allowing easy isolation of suppressors. At permissive temperatures (15° and 20°), a significant fraction of each mutant grew as nondauers.

The screens for suppressors of Daf-c mutations were carried out essentially as described (RIDDLE et al. 1981 ; THOMAS et al. 1993 ). EMS (ethyl methanesulfonate) mutagenesis was done as described (BRENNER 1974 ). X-rays were used at the dose of 2000–4000 rads. Mutagenized worms were placed on seeded 10-cm NG agar plates and allowed to lay eggs overnight. The plates were placed at the permissive temperature to allow the F₁ progeny to grow to nondauer adults. Once the F₁ progeny were past the dauer stage (L3, L4, or adults), the plates were shifted to the restrictive temperature. Three days after the shift, the plates were visually screened for nondauers (L4 or young adult), using a dissecting microscope. These nondauers were picked to the restrictive temperature to confirm that they carried heritable suppressor mutations.

The restrictive temperature of 25° was used for daf-14(m77) and daf-1(sa184). Although daf-14 is not completely penetrant at this temperature (typically 99% dauers), picked nondauers were significantly enriched for revertants. For daf-8-(sa234), 26° was used as the restrictive temperature, since at 25° the penetrance was too low to efficiently isolate suppressors.

The mutations we isolated varied significantly for their strength of suppression. Because of difficulties associated with analyzing low penetrance alleles, and because these were more likely to be nonspecific suppressors (see below), only strong suppressors were kept and analyzed. The cutoff was arbitrarily set at 90% suppression (<10% dauers). However, this is not a well-defined limit, because of the assay-to-assay variability in the frequency of dauer formation. The difference in strengths of the Daf-c mutations used [daf-1(sa184) > daf-14(m77) > daf-8(sa234)] probably also made this cutoff effectively different for each screen. In practice, however, types of mutations obtained from each screen were not obviously different.

sa756 was found as a spontaneous non-Daf-c revertant in the unc-24 daf-14(m77) background by M. Ailion. This mutation was analyzed in parallel with the suppressors generated by mutagenesis. Two additional alleles of scd-1, mg94 and mg99, were provided by G. Patterson and G. Ruvkun. The scd-2 (sa935) allele was provided by M. Ailion.

Nonspecific suppression of group 2 Daf-c genes:
Previous empirical observations suggested that most mutations used as genetic markers partially suppress the Daf-c phenotype of many mutants. To quantitate this effect, several daf-14(m77); marker double mutants were assayed. Mutations in dpy-5, rol-6, unc-32, dpy-11, unc-22, and unc-44 significantly suppressed the Daf-c phenotype. With the exception of unc-44, the nonspecific suppression ranged from 17 to 45%. Thus, the strong suppression (often >90% suppressed) observed for scd mutations analyzed here is likely to be caused by a more specific interaction. It is not clear why marker mutations, some in genes encoding cuticular collagens, suppress the Daf-c phenotype. The unc-44(e362) mutation we assayed strongly suppressed group 2 Daf-c mutants, perhaps indicating a specific interaction.

Mapping and complementation tests:
Suppressors were mapped and complementation tested using the suppression phenotype. For scd-2 and scd-3, complementation tests using single mutant phenotypes (dauer pheromone insensitivity and abnormal male tail morphology, respectively) were also done and gave consistent results. To assign mutations to previously described genes, complementation tests against daf-3(e1376), daf-5(sa205), daf-5(e1385), and daf-12(m20) were done. Complementation tests against scd-2(sa935) (M. AILION, personal communication) were also used to assign alleles to that complementation group. For all scd alleles presented here, the mutation was mapped to a chromosome before complementation tests were done. Thus at least two independent pieces of experimental data confirm the assignment of each allele to a scd gene.

The following type of complementation test was used most frequently to assign a mutation to a scd gene. daf-c; scd-A males were mated to marked daf-c; scd-B hermaphrodites and the unmarked progeny assayed at 25° for frequencies of dauers and nondauers. For scd-3 alleles, daf-c; scd/+ males were used, since scd-3 males do not mate (data not shown).

To test for linkage to the X chromosome and for dominance, daf-c males were crossed to daf-c; scd hermaphrodites, and the progeny were assayed at 25°. X-linkage was indicated by the presence of daf-c; scd/0 nondauer males and daf-c; scd/+ dauer hermaphrodites. Dominance was indicated by the presence of daf-c; scd/+ nondauer males and nondauer hermaphrodites (and by the absence of dauers, since hermaphrodite self-progeny are also nondauers). Autosomal linkage was indicated by the presence of daf-c; scd/+ dauers (and the absence of male nondauers).

Suppressor mutations were mapped to chromosomes as follows. At 25°, nondauer progeny of daf-c; scd/mk (or scd/+ mk/+) were picked (mk, marker). These were placed individually onto separate plates at 25° and allowed to self. The progeny were first scored to confirm that the suppressor was homozygous and then scored for segregation of the mk mutation. Confirmation of the suppressor genotype was necessary because of the incomplete penetrance of the Daf-c mutations. If the suppressor mutation was not linked to the marker, two-thirds of the nondauers segregated the marker mutation. If the suppressor mutation was linked, very few sup scd homozygotes segregated the marker mutation. Various methods were used to map the suppressor mutations to specific intervals on chromosomes. Methods, as well as results, are summarized in Table 3.

The following data argue that scd-1, scd-2, and scd-3 are new genes. scd-1(sa248) maps to the mes-1 unc-9 interval on chromosome X and complements daf-3 and daf-12. A similar map position (within the egl-15 unc-9 interval) was obtained independently for scd-1(sa318) (data not shown). scd-2(sa249) was mapped to the dpy-11 rol-3 interval of chromosome V. Furthermore, an independently isolated allele scd-2(sa935) was mapped to the dpy-11 unc-70 interval (M. AILION, personal communication). scd-3(sa253) and scd-3(sa246) were mapped to the unc-11 dpy-5 interval on chromosome I. No previously described genes affecting dauer formation map to these intervals. For scd-3, complementation tests were carried out against Mab genes lin-44 and egl-34 in the same interval. scd-3 complemented mutations in both of these genes.

Assays for Daf-c and Daf-d phenotypes:
Starvation assays were done to test for the ability to form dauers under strong dauer-inducing conditions (VOWELS and THOMAS 1992 ). One to four animals of each genotype were placed on seeded 5-cm NG agar plates and allowed to starve at 25°. Four to 6 days after the food was exhausted, the plates were flooded with 1% sodium dodecyl sulfate (SDS) solution to select for dauers. (Dauers are resistant to SDS; CASSADA and RUSSELL 1975 .) After 10–15 min, the plates were scored visually for dauers under the dissecting microscope. For scd-3 alleles, two to eight parents per plate were used because of low brood size.

Assays of dauer formation efficiency under high concentration of dauer pheromone (pheromone plate assay) were done essentially as described (GOLDEN and RIDDLE 1984B ). Briefly, synchronous populations of worms were grown on plates supplemented with crude dauer pheromone preparation. Food was provided as a thick suspension of Escherichia coli placed on the agar media, which contained streptomycin and lacked peptone to inhibit bacterial growth.

Frequencies of dauers formed by daf-c; scd double mutants under normal growth conditions were measured essentially as described (VOWELS and THOMAS 1992 ). Typically, 10 adult animals were allowed to lay eggs on seeded 5-cm NG plates for 6 hr at room temperature (23°). The parents were then removed and the plates were placed at 25°. The numbers of dauers and nondauers were scored 50 hr later. For some slow growing strains, the plates were scored at 72 hr (3 days). Formation of pdk-1(lf) type partial dauers (Table 9) was also scored at 72 hr, since this made it easier to distinguish them from true dauers. Although some fluctuations in the incubator temperature were inevitable, most of the assays were done between 25.0° and 25.5°.

Other phenotypes:
Egl (egg-laying defect and retention of eggs) and Din (dark intestine) phenotypes were scored visually. To score the Cpy (clumping behavior) phenotype, three parent worms were placed on seeded 5-cm NG agar plates and left undisturbed for 3 days at 20°. The distribution of the progeny on the plate was observed using a dissecting microscope. Typically, two to four replicate plates of each strain were scored. The Mab phenotype and the gonad position defect of scd-3 were assayed using Nomarski optics. Brood sizes were counted by transferring the parent animal from one plate to another until animals stopped laying eggs or died. The partial dauer phenotype was scored under Nomarski optics. Life spans of mutants and the wild type were measured as described. Life spans of scd-1(sa248), scd-2(sa249), and sa315 were wild type (data not shown). The life span of daf-2(e1370); sa315 was not distinguishable from daf-2(e1370).

sa315 dosage analysis and sequencing:
The following results suggest that sa315 is recessive. First, in a mapping cross, nondauer progeny of daf-1; sa315/+ were picked and the genotype confirmed in the next generation. All nondauers picked were sa315/sa315, suggesting that most sa315/+ animals were dauers. Second, sa315 homozygous males were crossed to unc-4 hermaphrodites and progeny grown on pheromone plates. Many non-Unc dauers were observed, indicating that sa315/+ animals are capable of forming dauers, unlike sa315/sa315 homozygotes, which are Daf-d.

To test the phenotype of sa315/Df, progeny of sa315/mnDf89 parents were grown on pheromone plates. From one set of assays, 19 were dauers or partial dauers and 116 were nondauers. Since sa315 homozygotes assayed in parallel did not form any dauers or partial dauers (n = 140), this suggests that sa315/Df is less Daf-d than is sa315/sa315, which would indicate that sa315 is a gain-of-function mutation. Curiously, most of the dauers appeared to be partial dauers.

Genomic fragments containing age-1 exons were amplified by PCR, using purified total genomic DNA of an sa315-bearing strain as the template. The bulk PCR product was sequenced and the result was compared with the reported age-1 mRNA sequence (MORRIS et al. 1996 ) as well as sequences from the genome project and an EST database.

	RESULTS AND DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Screens for suppressors of daf-14, daf-8, and daf-1:
To identify genes that interact with group 2 Daf-c genes daf-1, daf-8, and daf-14, we isolated mutations that suppressed the Daf-c phenotype of daf-1(sa184), daf-8(sa234), and daf-14(m77) (Table 1; MATERIALS AND METHODS). Both X-ray and EMS mutagenesis were used. In addition, a single suppressor allele (sa756) was found as a spontaneous mutation in daf-14(m77) background (M. AILION, personal communication).

Each screen produced suppressor mutations with a wide range of suppression strength. For example, in the case of the daf-14(m77) EMS screen, the level of suppression varied from 20 to 100% depending upon the suppressor mutation (data not shown). Because of the potential difficulty in analyzing weak suppressor mutations and also to avoid nonspecific suppressors, weak mutations were not kept for analysis (MATERIALS AND METHODS). In total, the 36 strongest suppressor mutations were kept for analysis. Although the screens were capable of isolating both recessive and dominant suppressors, all alleles were recessive (data not shown).

Complementation groups:
Mapping and complementation tests were carried out to place suppressor mutations into complementation groups (MATERIALS AND METHODS, Table 2, Table 3, and Fig 1). As expected, some suppressors were alleles of the previously described Daf-d genes, daf-3, daf-5, and daf-12 (RIDDLE et al. 1981 ; THOMAS 1993 ; THOMAS et al. 1993 ). In addition, four other complementation groups were identified. Three complementation groups correspond to previously undescribed genes, which we named scd-1, scd-2, and scd-3 (scd, suppressor of constitutive dauer). The fourth complementation group is represented by a single allele, sa315, which maps to the same genetic interval as the age-1 gene. As we have not determined whether sa315 defines a new gene or is an unusual allele of age-1, we have not given it a gene name.

View larger version (16K): In this window In a new window Download PPT slide   Figure 1. Chromosomal locations of scd genes.

With the exception of sa315, multiple alleles of each complementation group were found. This result suggests that most genes that mutate to cause strong suppression of the group 2 Daf-c mutations were identified. The spectrum of mutations obtained in each screen appears similar, despite the use of three different Daf-c alleles. daf-5, daf-12, and scd-1 alleles were obtained as suppressors of all three Daf-c mutations, and scd-3 and daf-3 alleles were isolated in two out of three. Similarly, subsequent tests showed that mutations in all three scd genes suppressed all group 2 Daf-c mutations (Table 5). However, as comparatively weaker suppressor mutations [e.g., scd-2(sa303)] did not strongly suppress the stronger Daf-c mutations [e.g., daf-1(sa184)], there was likely a bias toward stronger suppressors in screens carried out using daf-1(sa184).

scd single mutant phenotype:
The effect of scd mutations on dauer formation was tested using two assays, the starvation assay and the pheromone plate assay (Table 4 and MATERIALS AND METHODS; GOLDEN and RIDDLE 1984B ; VOWELS and THOMAS 1992 ). In the starvation assay, dauer formation is induced by allowing plates of worms to starve naturally. This causes a very strong dauer induction as a result of overcrowding and starvation. Strains carrying strong alleles of previously described Daf-d genes, daf-3, daf-5, and daf-12, formed very few dauers under this condition. In contrast, dauer formation by the scd-1 single mutant was indistinguishable from the wild type. scd-2 and scd-3 single mutant strains also formed dauers under this condition, although less efficiently than the wild type. The other assay used is a pheromone plate assay (Table 4), in which dauers are induced by the addition of crudely purified dauer pheromone to the media. With this assay, quantitative measurement of dauer frequency is possible, allowing detection of subtler differences in sensitivity to dauer-inducing stimuli. scd-2 and scd-3 single mutant strains formed very few dauers in this assay. In contrast, all five alleles of scd-1 tested (sa248, sa317, sa318, mg94, and mg99) had dauer pheromone responses indistinguishable from the wild type.

daf-c; scd double mutants:
To test the generality of suppression of group 2 Daf-c mutations, scd mutations were constructed in combination with daf-1, daf-7, daf-8, and daf-14. In general, the scd mutations suppressed all group 2 Daf-c mutations (Table 5). The suppression was incomplete in all cases, and the level of suppression varied according to the strengths of the alleles. For example, daf-1(sa184) and daf-7(e1372) produce a stronger Daf-c phenotype than do daf-8(sa234) and daf-14(m77). Accordingly, daf-1; sa249 and daf-7; sa249 formed significantly more dauers than daf-8; sa249 and daf-14; sa249. This incompleteness of suppression is not due to the choice of scd alleles we tested. From our screens, all isolated alleles that completely suppressed were alleles of daf-3, daf-5, or daf-12 (sa244, sa312, sa323, sa327, sa328, sa756, sa793, sa319, sa324, sa325, sa251, sa314, sa322, sa737, and sa796; data not shown). The weak phenotype of the scd alleles probably also accounts for the lack of scd alleles from earlier group 2 Daf-c suppressor screens, which were carried out using the strong Daf-c mutations daf-7(e1372) and daf-4(e1364) (RIDDLE et al. 1981 ; THOMAS et al. 1993 ).

In addition to dauer formation, the suppression of the pleiotropic phenotypes caused by the group 2 Daf-c mutation daf-14(m77) was scored (Table 6; TRENT et al. 1983 ; THOMAS et al. 1993 ). scd-1 alleles suppressed all of the pleiotropies, although the suppression was partial for some phenotypes. scd-2 alleles did not exhibit any effect on the pleiotropies. However, given the weaker Daf-c suppression phenotype of the scd-2 alleles in comparison to the scd-1 alleles (Table 5), the possibility that scd-2 alleles weakly affect the pleiotropies cannot be ruled out. scd-3 alleles suppressed the Cpy phenotype but not the Din phenotype. Although this may indicate a phenotype-specific interaction, suppression of the Cpy phenotype may also be a result of an unrelated pleiotropic sensory or motor defect of scd-3.

Placement of scd genes in the dauer pathway:
To further place the scd genes in the dauer pathway, double mutants were also built between scd mutations and daf-c mutations from the group 1 pathway (daf-11) and the daf-2/age-1 (daf-2) pathways (Table 5). Mutations in scd-1 and scd-2 suppressed group 2 Daf-c mutations but not daf-11(sa195) or daf-2(e1370), tentatively placing scd-1 and scd-2 in the group 2 (DAF-7/TGF-ß) pathway. For scd-1, this placement is further supported by the suppression of group 2 pleiotropies (Egl, Din, and Cpy). Like daf-3 (Smad) mutations, in which these pleiotropies are also suppressed (THOMAS et al. 1993 ; PATTERSON et al. 1997 ), scd-1 is likely to encode a component of the DAF-7/TGF-ß signaling pathway. scd-2 mutations do not suppress the pleiotropies, but have a stronger Daf-d phenotype than do scd-1 alleles. Thus, scd-2 probably functions at a different point in the dauer pathway from scd-1.

scd-3 mutants suppressed group 2 Daf-c mutants and daf-11(sa195) but not daf-2(e1370). Strong daf-3 and daf-5 mutations partially suppress daf-11 mutants. Unlike those genes, the level of suppression by scd-3 appears comparable for both daf-11 and group 2 Daf-c mutations, suggesting that scd-3 functions downstream of both.

Pleiotropic phenotypes of scd-3 mutants:
Uniquely among genes identified here, scd-3 alleles exhibited multiple pleiotropic phenotypes not obviously related to their dauer phenotype. The same pleiotropies were observed for three independently isolated alleles, sa253, sa320, and sa795, and are thus caused by mutations in the scd-3 gene. The fourth scd-3 allele, sa246, fails to complement the other scd-3 alleles for the suppressor phenotype, but did not exhibit some of the pleiotropies (Table 2). The pleiotropies include the following:

1. Low brood size: sa253, sa320, and sa795 reduced the hermaphrodite brood size (Table 7). As indicated by the large standard deviation, the phenotype was quite variable and individual brood sizes ranged from 0 to >100.

2. Egg-laying defect (Egl): Strains carrying any of the four scd-3 alleles retained eggs in their gonad and became bloated. Often, eggs hatched inside the parent and formed a "bag of worms," killing the parent. This probably contributes to the low brood size, although it is not the sole cause, since some animals that did not bag also had reduced brood size.

3. Gonadal L/R reversal: In the bilobed gonad of the wild-type C. elegans, the anterior half of the gonad is located on the right side of the intestine whereas the posterior half is located on the left side. In strains carrying the scd-3 alleles sa253, sa320, or sa795, the left/right positions of the gonad halves were frequently reversed (Table 8). The gonad position of the sa246 mutant was also affected, although less frequently. Anterior/posterior and dorsal/ventral organizations of each gonad arm were normal in most individuals. However, occasionally a severely misplaced or abnormally shaped gonad was observed ("other" in Table 8). The reversal of the posterior half of the gonad was the most frequent, occurring in 30 out of 75 animals (data for three strong alleles combined).

4. Weak Dpy (dumpy): All four scd-3 mutant alleles caused a variable and weak short body shape phenotype similar to weak Dpy mutants.

5. Male abnormal (Mab) phenotype: sa253, sa320, and sa795 mutant males exhibited abnormal tail morphology (Fig 2). Defects included deformation of the spicule, absence of the hook, and abnormal sensory rays. Defects in the sensory rays included missing rays, fused rays, and abnormally shaped rays, and were variable from animal to animal. All rays (one to nine) were affected to some extent. The combination of short body shape and male abnormality is also caused by mutations in the Sma/Mab pathway, a separate TGF-ß-related signaling pathway (SAVAGE et al. 1996 ). However, most Sma/Mab pathway mutants specifically affect rays four, five, six, and seven, and the defects are milder than those observed for scd-3. Therefore, scd-3 probably does not affect the Sma/Mab pathway.

   
   

   View larger version (72K): In this window In a new window Download PPT slide   Figure 2. The Mab phenotype of scd-3. Left, the tail of a phenotypically wild-type C. elegans male [scd-3(sa795)/+] showing the sensory rays. Eight rays are visible on either side. Right, the tail of a typical scd-3(sa253) male. The defect caused by the scd-3 mutation is variable and includes missing rays, fused rays (not shown), and deformed rays (open arrowhead). Other structures in the male tail, including the hook and spicules, are also affected (not shown).

sa315 affects the age-1/daf-2 pathway:
The last complementation group is represented by a single allele sa315, originally isolated as a suppressor of daf-1(sa184). sa315 affected dauer formation of group 1 and group 2 Daf-c mutants in two ways (Table 9). First, the frequency of dauer formation was reduced. Second, any dauers that formed were partial dauers (defined as animals mosaic for dauer and nondauer phenotypes; Fig 3 and MATERIALS AND METHODS). Formation of partial dauers is a hallmark of mutations in the age-1/daf-2 pathway, as partial dauers are not made by the wildtype or by group 1 and group 2 mutants under any condition (VOWELS and THOMAS 1992 ). Partial dauers formed by group 2 daf-c; sa315 double mutants had dauer alae but the pharynx was not remodeled to a dauer-like state. This combination of phenotypes and the overall appearance of the partial dauers (thin but pale) were similar to those of group 2 daf-c; daf-16 and group 2 daf-c; daf-18 double mutants (VOWELS and THOMAS 1992 ), suggesting that the sa315 mutation caused a defect similar to daf-16(lf) and daf-18(lf). In general, daf-c; sa315 double mutants formed fewer partial dauers than daf-c; daf-16 and daf-c; daf-18 (Table 9); however, this difference may be merely quantitative.

View larger version (142K): In this window In a new window Download PPT slide   Figure 3. sa315 partial dauer phenotype. Shown are the dauer alae (or lack thereof; A, C, E, G, and I) and the pharynx (B, D, F, H, and J) from dauers, partial dauers, and an L3. The scale bar is 0.1 mm. (A and B) a daf-7(e1372) dauer. (C and D) a daf-7; sa315 partial dauer. (E and F) a daf-7; daf-16(m27) partial dauer. (G and H) a daf-7; akt-1(mg144) dauer. (I and J) wild-type L3 larva. In a dauer, the dauer alae are present (A and G) and the pharynx is narrower and more tapered (B and H). In an L3 larva, the alae are absent (I) and the pharynx is wide (J). In partial dauers, the alae are present (C and E) but the pharynx shape is closer to that of the wild-type L3 (D and F).

sa315 also interacted with Daf-c mutations in the age-1/ daf-2 pathway (Table 9). First, sa315 suppressed the Daf-c phenotype of pdk-1(sa680), as do daf-16(lf) and akt-1(d). Second, sa315 interacted with daf-2. The daf-2 (e1370) single mutant formed normal dauers only, whereas the daf-2(e1370); sa315 double mutant formed partial dauers that were dark and thick (different from group 2 daf-c; sa315 partial dauers). These partial dauers resembled partial dauers made by the daf-2; akt-1 (mg144dm) double mutant. In contrast, daf-2; daf-16(lf) and daf-2; daf-18(lf) double mutants formed partial dauers like group 2 daf-c; sa315 mutants. Therefore, sa315 differs from daf-16(lf) and daf-18(lf) in its interaction with daf-2. Although the pattern of interactions does not exactly match that of any previously identified mutants, sa315 does appear to affect the daf-2/age-1 pathway.

The similarity between daf-2; sa315 and daf-2; akt-1(d) led us to test the interaction of akt-1(d) and pdk-1(d) with group 2 Daf-c alleles (Table 9). Unlike sa315, daf-16(lf), and daf-18(lf), the akt-1 and pdk-1 alleles did not show any obvious effect on dauer formation by daf-7(e1372) or daf-14(m77). Closer examination of the dauers form- ed by the daf-7; akt-1(d) double mutant indicated that the dauers were not partial (Fig 3).

The nature of the sa315 mutation:
Genetic mapping placed the sa315 mutation in the same region of chromosome II as age-1. Since age-1(null) is Daf-c, this suggested the possibility that sa315 is a gain-of-function mutation in age-1. The fact that only one sa315-like allele was found among the 36 mutations analyzed in this study is also consistent with this idea, since gain-of-function mutations are typically rare. Although sa315 is recessive, a preliminary gene dosage analysis of sa315 suggested that sa315 is a gain-of-function mutation (MATERIALS AND METHODS). However, sequencing of the entire age-1 coding region from the sa315 mutant detected no mutations (MATERIALS AND METHODS). Therefore, sa315 may be a mutation in the noncoding region of age-1 or an allele of a novel gene in the region.

	ACKNOWLEDGMENTS

We thank M. Ailion for isolation and analysis of daf-5(sa756) and scd-2(sa935). We thank G. Ruvkun and members of his lab including G. Patterson for scd-1 alleles and C. Wolkow for age-1 sequencing reagents. Some C. elegans strains used in this work were provided by the Caenorhabditis Genetics Center (CGC), which is funded by the National Institutes of Health Center for Research Resources (NCRR).

Manuscript received March 31, 2000; Accepted for publication June 15, 2000.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

BARGMANN, C. I. and H. R. HORVITZ, 1991  Control of larval development by chemosensory neurons in Caenorhabditis elegans.. Science 251:1243-1246[Abstract/Free Full Text].

BIRNBY, D., E. A. MALONE, J. J. VOWELS, H. TIAN, and P. COLACURCIO et al., 2000  A transmembrane guanylyl cyclase (DAF-11) and Hsp-90 (DAF-21) regulate a common set of chemosensory behaviors in C. elegans.. Genetics 155:85-104[Abstract/Free Full Text].

BRENNER, S., 1974  The genetics of Caenorhabditis elegans.. Genetics 77:71-94[Abstract/Free Full Text].

CASSADA, R. C. and R. L. RUSSELL, 1975  The dauer larva, a post-embryonic developmental variant of the nematode Caenorhabditis elegans.. Dev. Biol. 46:326-342[Medline].

ESTEVEZ, M., L. ATTISANO, J. L. WRANA, P. S. ALBERT, and J. MASSAGUE et al., 1993  The daf-4 gene encodes a bone morphogenetic protein receptor controlling C. elegans dauer larva development. Nature 365:644-649[Medline].

GEORGI, L. L., P. S. ALBERT, and D. L. RIDDLE, 1990  daf-1, a C. elegans gene controlling dauer larva development, encodes a novel receptor protein kinase. Cell 61:635-645[Medline].

GIL, E. B., E. M. LINK, L. X. LIU, C. D. JOHNSON, and J. A. LEES, 1999  Regulation of the insulin-like developmental pathway of Caenorhabditis elegans by a homolog of the PTEN tumor suppressor gene. Proc. Natl. Acad. Sci. USA 96:2925-2930[Abstract/Free Full Text].

GOLDEN, J. W. and D. L. RIDDLE, 1982  A pheromone influences larval development in the nematode Caenorhabditis elegans.. Science 218:578-580[Abstract/Free Full Text].

GOLDEN, J. W. and D. L. RIDDLE, 1984a  The Caenorhabditis elegans dauer larva: developmental effects of pheromone, food, and temperature. Dev. Biol. 102:368-378[Medline].

GOLDEN, J. W. and D. L. RIDDLE, 1984b  A pheromone-induced developmental switch in Caenorhabditis elegans: temperature-sensitive mutants reveal a wild-type temperature-dependent process. Proc. Natl. Acad. Sci. USA 81:819-823[Abstract/Free Full Text].

GOTTLIEB, S. and G. RUVKUN, 1994  daf-2, daf-16 and daf-23: genetically interacting genes controlling dauer formation in Caenorhabditis elegans.. Genetics 137:107-120[Abstract].

HORVITZ, H. R., S. BRENNER, J. HODGKIN, and R. K. HERMAN, 1979  A uniform genetic nomenclature for the nematode Caenorhabditis elegans.. Mol. Gen. Genet. 175:129-133[Medline].

INOUE, T. and J. H. THOMAS, 2000  Targets of TGF-beta signaling in Caenorhabditis elegans dauer formation. Dev. Biol. 217:192-204[Medline].

KIMURA, K. D., H. A. TISSENBAUM, Y. LIU, and G. RUVKUN, 1997  daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans.. Science 277:942-946[Abstract/Free Full Text].

LIN, K., J. B. DORMAN, A. RODAN, and C. KENYON, 1997  daf-16: an HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans.. Science 278:1319-1322[Abstract/Free Full Text].

MALONE, E. A. and J. H. THOMAS, 1994  A screen for nonconditional dauer-constitutive mutations in Caenorhabditis elegans.. Genetics 136:879-886[Abstract].

MALONE, E. A., T. INOUE, and J. H. THOMAS, 1996  Genetic analysis of the roles of daf-28 and age-1 in regulating Caenorhabditis elegans dauer formation. Genetics 143:1193-1205[Abstract].

MASSAGUE, J., 1998  TGF-beta signal transduction. Annu. Rev. Biochem. 67:753-791[Medline].

MIHAYLOVA, V. T., C. Z. BORLAND, L. MANJARREZ, M. J. STERN, and H. SUN, 1999  The PTEN tumor suppressor homolog in Caenorhabditis elegans regulates longevity and dauer formation in an insulin receptor-like signaling pathway. Proc. Natl. Acad. Sci. USA 96:7427-7432[Abstract/Free Full Text].

MORRIS, J. Z., H. A. TISSENBAUM, and G. RUVKUN, 1996  A phosphatidylinositol-3-OH kinase family member regulating longevity and diapause in Caenorhabditis elegans.. Nature 382:536-539[Medline].

OGG, S. and G. RUVKUN, 1998  The C. elegans PTEN homolog, DAF-18, acts in the insulin receptor-like metabolic signaling pathway. Mol. Cell 2:887-893[Medline].

OGG, S., S. PARADIS, S. GOTTLIEB, G. I. PATTERSON, and L. LEE et al., 1997  The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans.. Nature 389:994-999[Medline].

PARADIS, S. and G. RUVKUN, 1998  Caenorhabditis elegans Akt/PKB transduces insulin receptor-like signals from AGE-1 PI3 kinase to the DAF-16 transcription factor. Genes Dev. 12:2488-2498[Abstract/Free Full Text].

PARADIS, S., M. AILION, A. TOKER, J. H. THOMAS, and G. RUVKUN, 1999  A PDK1 homolog is necessary and sufficient to transduce AGE-1 PI3 kinase signals that regulate diapause in Caenorhabditis elegans.. Genes Dev. 13:1438-1452[Abstract/Free Full Text].

PATTERSON, G. I., A. KOWEEK, A. WONG, Y. LIU, and G. RUVKUN, 1997  The DAF-3 Smad protein antagonizes TGF-beta-related receptor signaling in the Caenorhabditis elegans dauer pathway. Genes Dev. 11:2679-2690[Abstract/Free Full Text].

REN, P., C. S. LIM, R. JOHNSEN, P. S. ALBERT, and D. PILGRIM et al., 1996  Control of C. elegans larval development by neuronal expression of a TGF-beta homolog. Science 274:1389-1391[Abstract/Free Full Text].

RIDDLE, D. L., and P. S. ALBERT, 1997 Genetic and environmental regulation of dauer larva development, pp. 739–768 in C. elegans II, edited by D. L. RIDDLE, T. BLUMENTHAL, B. J. MEYER and J. R. PRIESS. Cold Spring Harbor Laboratory Press, Plainview, NY.

RIDDLE, D. L., M. M. SWANSON, and P. S. ALBERT, 1981  Interacting genes in nematode dauer larva formation. Nature 290:668-671[Medline].

ROUAULT, J. P., P. E. KUWABARA, O. M. SINILNIKOVA, L. DURET, and D. THIERRY-MIEG et al., 1999  Regulation of dauer larva development in Caenorhabditis elegans by daf-18, a homologue of the tumour suppressor PTEN. Curr. Biol. 9:329-332[Medline].

SAVAGE, C., P. DAS, A. L. FINELLI, S. R. TOWNSEND, and C. Y. SUN et al., 1996  Caenorhabditis elegans genes sma-2, sma-3, and sma-4 define a conserved family of transforming growth factor beta pathway components. Proc. Natl. Acad. Sci. USA 93:790-794[Abstract/Free Full Text].

SCHACKWITZ, W. S., T. INOUE, and J. H. THOMAS, 1996  Chemosensory neurons function in parallel to mediate a pheromone response in C. elegans.. Neuron 17:719-728[Medline].

THATCHER, J. D., C. HAUN, and P. G. OKKEMA, 1999  The DAF-3 Smad binds DNA and represses gene expression in the Caenorhabditis elegans pharynx. Development 126:97-107[Abstract].

THOMAS, J. H., 1993  Chemosensory regulation of development in C. elegans.. Bioessays 15:791-797[Medline].

THOMAS, J. H., D. A. BIRNBY, and J. J. VOWELS, 1993  Evidence for parallel processing of sensory information controlling dauer formation in Caenorhabditis elegans.. Genetics 134:1105-1117[Abstract].

TRENT, C., N. TSUNG, and H. R. HORVITZ, 1983  Egg-laying defective mutants of the nematode C. elegans.. Genetics 104:619-647[Abstract/Free Full Text].

VOWELS, J. J. and J. H. THOMAS, 1992  Genetic analysis of chemosensory control of dauer formation in Caenorhabditis elegans.. Genetics 130:105-123[Abstract].

YEH, W., 1991 Genes acting late in the signaling pathway for Caenorhabditis elegans dauer larval development. Thesis, University of Missouri, Columbia, MO.

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