switching in the nematode Caenorhabditis elegans

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 12388-12393, October 1996 Genetics Environmental induction and genetic control of surface antigen switching in the nematode Caenorhabditis elegans (nematode cuticle/immune evasion/signal transduction) DAVID G. GRENACHE*t, IAN CALDICOTTt, PATRICE S. ALBERTt, DONALD L. RIDDLEt, AND SAMUEL M. POLITZ*1 *Department of Biology and Biotechnology, Worcester Polytechnic Institute, 100 Institute Road, Worcester, MA 01609; and tdivision of Biological Sciences, University of Missouri, Columbia, MO 65211 Communicated by William B. Wood, University of Colorado, Boulder, CO, August 8, 1996 (received for review February 21, 1996) ABSTRACT Nematodes can alter their surface coat protein compositions at the molts between developmental stages or in response to environmental changes; such surface alterations may enable parasitic nematodes to evade host immune defenses during the course of infection. Surface antigen switching mechanisms are presently unknown. In a genetic study of surface antigen switching, we have used a monoclonal antibody, M37, that recognizes a surface antigen on the first larval stage of the free-living nematode Caenorhabditis elegans. We demonstrate that wild-type C. elegans can be induced to display the M37 antigen on a later larval stage by altering the growth conditions. Mutations that result in nonconditional display of this antigen on all four larval stages fall into two classes. One class defines the new gene srf-6 II. The other mutations are in previously identified dauer-constitutive genes involved in transducing environmental signals that modulate formation of the dauer larva, a developmentally arrested dispersal stage. Although surface antigen switching is affected by some of the genes that control dauer formation, these two processes can be blocked separately by specific mutations or induced separately by environmental factors. Based on these results, the mechanisms of nematode surface antigen switching can now be investigated directly. All nematodes have five postembryonic stages (Li through L4 and adult) separated by four molts. At each molt a new multi-layered cuticle is synthesized (1). The internal cuticle layers, composed primarily of collagens (2, 3), are covered by an insoluble matrix (2, 4) and the osmiophilic epicuticle (5), whose compositions are not well known. Outside of these layers is a 5-20-nm-thick surface coat composed primarily of glycoproteins (for review, see ref. 6). Unlike the other cuticle proteins, surface coat molecules are readily secreted into the environment (6). The molts allow abrupt, wholesale surface changes to occur when a new cuticle is made. Stage-specific surface differences are known to occur in a variety of nematode species, including the free-living species Caenorhabditis elegans (6-11). The potential importance of a stage-specific surface composition to parasitism is suggested by Trichinella spiralis infections of mammals. Later stages that express different surface antigens escape the immune attack directed against the stages present early in infection (12). Surface composition can also change within a single stage, e.g., in response to a new host or host tissue, and surface molecules can be shed in response to binding of immune effector cells or antibodies (for reviews, see refs. 6-9). Surface antigen switching results in the restriction of particular surface molecules to a specific time or developmental stage. Elucidating the mechanism of surface antigen switching would not only help explain responses of the nematodes to The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. 1734 solely to indicate this fact. 12388 external conditions, but also might allow formulation of a means to interfere with the immune evasion mechanisms of highly prevalent, medically important parasites (13, 14). Despite its potential significance, the genetic control of surface antigen expression in nematodes has not been investigated, except for several preliminary studies identifying C. elegans surface-altered mutants (10, 11, 15). One of these studies (11) forms the basis for the present work. We describe here an inducible surface antigen switch in C. elegans, using a monoclonal antibody (mab) probe that detects a surface antigen found on Li larvae grown under standard conditions. Under altered growth conditions, L2 larvae are induced to display this antigen. We investigated this surface antigen switch by isolating (11) and analyzing C. elegans mutants that display the Li surface antigen on stages L2 through L4 under all growth conditions. Some mutations affect only surface antigen switching, and define at least one new gene, while others also result in temperature-sensitive, constitutive formation of dauer larvae and are in previously described genes. The C. elegans dauer larva is a developmentally arrested dispersal stage (16) formed at the second molt, as an alternative to the L3, in response to high population density and limited food (17). The genes affecting both dauer larva formation and surface antigen switching normally act to prevent dauer larva formation in the presence of food, and their activities are required primarily at higher temperatures (e.g., 25 C) but not at 16 C (18). Hence, loss-of-function mutations in these genes result in a temperature-sensitive, dauerconstitutive (ts Daf-c) phenotype. These genes have been hypothesized to control neural transduction of environmental stimuli during dauer larva formation (19). Their involvement in surface antigen switching supports the idea that environmental stimuli also induce surface antigen alterations. However, we also show that surface antigen switching and dauer larva formation can be induced separately by specific environmental conditions or blocked separately by specific mutations, indicating that the two processes are controlled differently. MATERIALS AND METHODS Nematode Culture for Immunofluorescent Staining. Nematode stocks were grown at 16 C on nematode growth agar plates seeded w-ith Escherichia coli OP50 (20). For some assays, Abbreviations: Li, L2, L3, and L4, first, second, third, and fourth larval stages, respectively; mab, monoclonal antibody; ts, temperature sensitive; daf-c, Daf-c, dauer-constitutive; daf-d, Daf-d, dauerdefective; srf, Srf, surface antigen expression; CLD, constitutive larval display; ILD, inducible larval display. tpresent address: Department of Clinical Laboratory Science, Fitchburg State College, Fitchburg, MA 01420. Present address: Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721. ITo whom reprint requests should be addressed.

Genetics: Grenache et al. a few gravid nematodes were placed on plates and allowed to lay eggs for 2-3 h. Parents were then removed and progeny were grown synchronously at the temperatures indicated in Results. Pheromone Plates. In some studies, nematodes were grown on plates containing extracts of depleted culture medium known to contain dauer-inducing pheromone activity (21). Crude pheromone extract and a column-purified extract were prepared as described (22). For the latter, pheromone activity was eluted from a silicic acid column with CHCl3:CH30H (2:1, vol/vol), dried down, redissolved in water, filter-sterilized, and stored at 4 C. Either crude or column-purified pheromone extract was added to autoclaved nematode growth agar medium (made without peptone) just before pouring into 35 x 10-mm Petri dishes (2 ml each). The agar was seeded with E. coli OP50 suspended in S medium (prepared as described in ref. 23) containing streptomycin sulfate (final concentration, 50,ug/ml of agar), and used the next day. Immunofluorescent Staining of Live Nematodes with mab M37. Isolation and characterization of mab M37 were described previously (11). Worms were stained and observed as described for mab M38 (11), except that M37 incubation time was 2.5 h, and 15,ul of fluorescein isothiocyanate-conjugated secondary antibody was used in a 1.5-h incubation. For photomicrography, worms were anesthetized with NaN3 as described previously (11) and wet mounted on 5% agarose pads. Strains. Standard C. elegans genetic nomenclature is used (24). Isolation of strains carrying sff-6(yj5), srf-6(yjl3), srf- 6(yjl5), srf-6(yj43), srf-6(yj41), daf-7(yj11), and daf-7(yj12) has been described (11), as has isolation of the sff-2(yj262) mutant (15). Other dauer formation mutants (19), and dauerconstitutive (daf-c); dauer-defective (daf-d) double mutant strain construction and genotype confirmation methods (25) were described previously. A srf-6(yjl3) unc-4(e120) II; daf-3(el376) X strain was obtained by mating sff-6 unc-4/+ + males with daf-3 hermaphrodites. Male offspring were mated with daf-3 hermaphrodites, and Unc F2 progeny were picked individually to establish the desired mutant strain. Presence of srf-6(yjl3) was verified by complementation testing with sff-6 males; sff-6 homozygosity was inferred from the mutant's highly penetrant binding of mab M37 at stages L2-L4. Homozygosity of daf-3, inferred by the construction method, was confirmed by the strain's dauerdefective phenotype when grown to starvation at 25 C and its suppression of the ts Daf-c phenotype of daf-7(el372) in all segregants of genetic crosses with daf-7(e1372); daf-3(el376). A sff-6(yjl3) unc-4(el20) II; daf-12(m20) X strain was constructed similarly. Mapping and Complementation Testing. Mapping crosses and stock growth were performed at 16 C using standard methods (20). Visible mutant phenotypes were used as selected markers, and M37 immunofluorescence at stages L2-L4 served as the unselected marker. The sif mutants were tested for linkage with a set of autosomal uncoordinated markers; dpy-l(el) III was used to establish linkage of daf-7(yjll) and daf-7(yj12). For three-factor mapping of srf-6, Dpy non-unc, and Unc non-dpy recombinant progeny of dpy-1o(el28) unc- 4(e120)/srf-6(yj13) hermaphrodites were picked; mixed stages of homozygous recombinant stocks were tested for M37 binding. Complementation with srf-6 was tested by mating Srf homozygous males with unc-4 srf-6(yjl3) II hermaphrodites. L2-L4 stage, non-unc cross progeny were tested for M37 immunofluorescence. For complementation testing of srf-6 with chromosome II deficiencies, srf-6(yjl3) +/srf-6(yjl3) unc- 4(e120) males were mated with unc-4(e120) Df/mnCl [dpy- 1O(el28) unc-52(e444)] hermaphrodites. L2-L4 Unc-4 progeny containing srf-6(yjl3) opposite the deficiency were screened for M37 immunofluorescence. Strains carrying yjli and yjl2 were tested for complementation of their ts Daf-c Proc. Natl. Acad. Sci. USA 93 (1996) 12389 phenotype with daf-1 (m40) dpy-13(e184) IV and daf-7(el372) dpy-l(el) unc-32(e189) III. RESULTS Genetic Analysis of Mutants. When tested on wild-type worms grown under standard Conditions, mab M37 and M38 bound to the surface of Li larvae but not to other stages (11). In previously described immunofluorescence screens, nine ethylmethane sulfonate-induced mutants (11) were isolated that bind mab M37 and M38 at stages L1-L4, but not as adults (referred to as constitutive larval display, or CLD). Seven of these mutants have now been analyzed genetically, using mab M37 surface immunofluorescence as a phenotypic marker. Five mutations, including the previously described yj43 (11), displayed no distinct phenotype other than CLD (e.g., Fig. 1 A and B), showed linkage to unc-4 II and were allelic; these mutations define the srf-6 gene. Three-factor crosses and deficiency mapping localized srf-6 just to the right of dpy-10 II (Fig. 2). Two other mutations,yjll andyjl2, resulted in formation of dauer larvae independent of food limitation when grown at 25 C, a phenotype characteristic of ts Daf-c mutants (19). These mutations showed linkage to dpy-1 III and were allelic with daf-7(el372) III (data not shown). CLD in ts Dauer-Constitutive Mutants and srf-6 at Permissive Temperature. Based on the ts Daf-c phenotype ofyjll and yjl2, we tested strains carrying ts daf-c mutations in daf-1, daf-2, daf-4, daf-7, daf-8, daf-li, and daf-14 (19) for CLD. Surprisingly, most of the ts Daf-c mutants showed CLD when FIG. 1. Indirect immunofluorescence of C. elegans mutant strains grown at 16 C and stained with mab M37. Larval stages 1-4, but not adults, fluoresce, except in G where the daf-5 mutation suppresses fluorescence at all stages except Li. Images were recorded on TMax 400 film. (A and B) srf-6(yjl3); (C and D) daf-i(m40); (E and F) daf-i (m40);daf-12(m20); (G and H) daf-1 (m40); daf-5(el386). A, C, E, and G show epifluorescence images. The same fields are shown as bright field images in B, D, F, and H, respectively. (Bar = 0.5 mm.) Arrowheads in H indicate the positions of Li larvae that show positive immunofluorescence in G.

12390 Genetics: Grenache et al asmapuzuts dpy-io srr-,6 Q-L n IDI /n-26 rol-6 I1 IIf., unc-4 IF l 7 --- FIG. 2. Partial genetic map of C. elegans chromosome II, showing the position of srf-6 determined from data presented here. In threefactor crosses, recombinant progeny of dpy-10 unc-4/srf-6 parents were scored for CLD. Five recombination events occurred between dpy-10 and srf-6, and 52 were between srf-6 and unc-4 (data pooled from crosses with srf-6 alleles yj43, yjl3, and yjls). Consistent with these results, srf-6(yjl3) failed to complement chromosome II deficiencies mndf3l and mndf3o, but complemented mndf6l, mndf68, and mndf88. The position of lin-26 is from ref. 26, positions of other markers are from ref. 27, and positions of deficiencies are from ref. 28. grown at 16 C (Table 1), a temperature at which a minimal fraction of the population is induced to form dauer larvae (17, 18, 29). With the exception of daf-2 mutants, all were like sif-6 and bound mab M37 at stages L1-L4, but not as adults (Table 1; e.g., Fig. 1 C and D). Mutants in five different daf-c genes showed CLD with high penetrance (87-100%), and strains carrying each of four different daf-li alleles displayed partial and more variable penetrance (15-76%) of CLD. All three daf-2 mutants tested appeared similar to wild-type, displaying the mab M37 epitope only on the Li surface, and none of the srf-6 mutants showed a ts Daf-c phenotype at 25 C, the standard restrictive temperature. Some ts Daf-c phenotypes are expressed only at 27.5 C (30). Four srf-6 mutants showed no ts Daf-c phenotype at 27.5 C. In the fifth, srf-6(yj41), 5% of animals grown from hatching at 27.5 C developed into dauerlike larvae, but these were incompletely transformed for certain dauer characteristics including a dark intestine and Table 1. mab M37 immunofluorescence staining of L2-L4 stage dauer-constitutive mutants grown at 160C Antigen positive/total Genotype scored Range (%) daf-1(m40) 107/107 100 daf-2(el370) 0/116 0 daf-2(el369) 1/188 0-1 daf-2(m41) 1/111 0-1 daf-4(m63) 130/132 98-100 daf-7(el 372) 44/44 100 daf-8(el393) 132/136 95-100 daf-11(m47) 204/347 43-76 daf-11(m87) 66/134 17-76 daf- 1(sa194) 53/126 15-58 daf- 1(sal95) 38/70 24-67 daf-14(m77) 153/162 87-100 Mixed stage nematode stocks were grown and stained as described in Materials and Methods. Entries combine results of two trials, or four trials for daf- 1(m47). In all cases, Li larvae were antigen-positive, and adults were antigen-negative. Proc. Natl. Acad. Sci. USA 93 (1996) remodeled pharynx, and continued to feed and develop into L4 larvae and adults at 27.5 C. The fact that ts Daf-c mutants show CLD at permissive temperature suggested that the ability to form dauer larvae might not be required for CLD. To test this possibility, we constructed double mutants carrying a ts daf-c mutation and a dauer-defective (daf-d) mutation, such as daf-12(m20). The daf-d mutations we used prevent dauer formation even in the presence of a ts daf-c mutation (19, 31) and do not themselves result in CLD (Table 2). All daf-c; daf-12 double mutants tested displayed CLD at 16 C (Table 2; e.g., Fig. 1 E and F). In contrast to the daf-12 results, all daf-c mutants carrying a daf-d mutation in either daf-3 or daf-5 showed M37 binding only at the Li stage (Table 2; e.g., Fig. 1 G and H), suggesting that the wild-type activities of daf-3 and daf-5 are both required for CLD. Because the double mutants with daf-3 or daf-5 bound M37 as Li larvae, the lack of antibody binding at stages L2-L4 appears to be an effect on surface antigen switching at the Li molt rather than a defect in antigen expression. A srf-6 mutation in combination with daf-d mutations exhibited a third pattern of genetic interaction; neither a daf-12 mutation nor a daf-3 mutation blocked CLD (Table 2). In contrast to the ts daf-c genes, therefore, srf-6 does not appear to interact with daf-3. CLD of daf-c Mutants at Restrictive Temperature. The results shown in Tables 1 and 2 were obtained at 16 C, a permissive temperature for the ts Daf-c phenotype (19). To test CLD under dauer-inducing conditions, ts daf-c strains were grown at 25 C and tested for mab M37 binding as L2 or dauer larvae. We refer to all animals that have undergone one molt since hatching as L2 larvae, and we do not attempt to distinguish L2 larvae from the L2d larvae that precede dauer formation (17). This distinction is not crucial here, because the formal definition of CLD requires only that the M37 epitope be displayed on larvae that have molted at least once. mab M37 binding by L2 and dauer larvae grown under conditions that induced nearly 100% dauer larvae is shown in Table 3. None of the dauer larvae tested displayed the M37 antigen. However, consistent with the results obtained at 16 C, L2 larvae of all daf-c strains tested, with the exception of daf-2(e1370), displayed the M37 antigen. A comparatively small fraction of daf-1l mutant L2 larvae stained. As shown below, M37 antigen display can also be induced in wild-type L2 larvae. Environmental Signals Modulate Larval Display of the M37 Antigen in Wild Type and Mutants. The decision between dauer and non-dauer development depends on the balance Table 2. mab M37 immunofluorescent staining of L2-L4 double mutant larvae grown at 160C Percent antigen-positive (total no. scored) daf-d mutant daf-c or sif daf-12 allele mutant daf-3 daf-5 m20 m116 + 0 (53) 0 (47) 0 (58) 0 (159) daf-1 0(107) 0(80) 98 (121) 77 (244) daf-4 0 (122) 0 (138) 99 (113) ND daf-7 0 (124) 0 (97) 99 (146) ND daf-8 0 (96) 0 (138) 79 (86) 67 (349) daf-li 0 (103) 0 (94) 24 (104) ND daf-14 0 (98) 0 (85) 92 (113) ND srf-6 95 (136) ND 86 (126) ND Results are sums of two experiments on mixed stage stocks, except for daf-12(m116), which was tested once. In all cases, Li larvae were antigen-positive, and adults were antigen-negative. Mutant alleles were daf-3(el376), daf-5(el386), daf-11(m47), daf-8(m85), daf- 4(el364), and srf-6(yjl3); alleles of other daf-c genes were those listed in Table 1.

Genetics: Grenache et al. Table 3. mab M37 immunofluorescence staining of daf-c L2 and dauer larvae grown at 25 C Percent antigen-positive (total no. scored) Dauer Dauer larvae Genotype L2 larvae larvae formed (%) daf-l (m40) 89 (28) 0 (51) 100 daf-2(el370) 0 (188) 0 (61) 100 daf-4(m63) 97 (38) 0 (61) 98 daf-4(m592) 80 (46) 0 (60) 100 daf-7(e1372) 58 (19) 0 (53) 100 daf-8(el393) 63 (82) 0 (77) ND daf-11(m87) 17 (29) 0 (30) 97 daf-11(sa195) 3 (33) 0 (19) 100 Worms were grown on nematode growth plates from eggs laid during a 3-h period. L2 larvae were harvested for staining after 40-48 h of growth, at which time a few animals on each plate were entering the L2-dauer larva molt. Dauer larvae were individually picked from 3-5-day-old plates and pooled for staining. Dauer formation was determined on parallel control plates containing at least 50 animals per plate. between dauer-inducing pheromone and an antagonistic food signal (17). The daf-c genes are believed to represent intermediate steps in a signaling pathway (19, 31, 32) involving processing of environmental cues by chemosensory neurons (33-36) and intercellular signal transduction. Indeed, daf-1 and daf-4 encode putative protein growth factor receptors (37, 38). The involvement of daf-c genes in surface antigen switching suggested that surface antigen expression might also be modulated by environmental signals, perhaps the same ones used for dauer formation. To test this, worms were grown from hatching to the L2 stage on plates containing limited food and an extract of liquid culture medium containing dauer-inducing pheromone activity (21) and then stained with M37. Table 4. mab M37 immunofluorescence of L2 larvae formed in the presence or absence of crude pheromone extract No. of Percent antigen-positive Genotype Extract trials (total no. scored) Wild-type dauer + 1 0 (19) Wild-type - 1 12 (94)*t Wild-type + 4 92 (495) daf-12(m20) - 1 16 (55)*t daf-12(m20) + 3 93 (311) daf-12(m1l6) + 3 91 (225) daf-12(m25) + 2 99 (141) daf-3(el376) + 2 87 (259)* daf-3(mlo) + 1 69 (157)* daf-5(el386) + 3 65 (160)* daf-7; daf-3 + 1 88 (246)* daf-1; daf-3 + 1 100(130)* daf-6(el377) + 1 81 (226) srf-2(yj262) + 1 0 (195) All samples were grown for 48 h at 15 C on agar media containing no peptone, 50,ul of crude pheromone extract (+ extract), or water (- extract) per 2-ml plate, and 0.5-0.6 mg of streptomycin-treated E. coli OP50 per plate. Alleles not listed were daf-l (m40), daf-3(e1376), and daf-7(e1372). Dauer formation in parallel wild-type + extract plates ranged from 82-99%. *Positively staining worms in these trials showed variable results, including partially and weakly staining individuals. twild-type or daf-d L2 larvae were antigen-positive in the absence of pheromone extract only when grown on the modified medium used in this Table; in genetic mapping tests, complementation tests, and the experiments of Tables 1-3, wild-type or daf-d L2-L4 larvae grown on NGM medium and non-streptomycin-treated bacteria were all antigen-negative. Proc. Natl. Acad. Sci. USA 93 (1996) 12391 Wild-type L2 larvae grown in the presence of crude extract bound mab M37 (71-99% in four trials, Table 4), indicating that display at a stage later than Li can occur even in wild-type worms. We refer to this inducible L2 display of the M37 epitope as inducible larval display (ILD). Wild-type dauer larvae grown under these conditions did not bind mab M37. L2 larvae grown on limited food without added extract showed a much lower level of ILD (12%, Table 4) and exhibited partial or weak staining (not shown). As observed with daf-c; daf-12 double mutants (Table 2), three different mutations in daf-12 did not interfere with ILD (Table 4). In contrast with daf-12, mutations in dauer-defective genes daf-3 or daf-5 partially interfered with ILD in L2 larvae grown in the presence of crude pheromone extract (Table 4). Although the majority of these mutant larvae showed mab M37 binding, often the staining pattern was partial or weak (data not shown). Frequently, only a portion of the body surface bound M37. Variable staining was obtained even when the genotype included a daf-1 or daf-7 ts daf-c allele in addition to a daf-3 dauer-defective mutation. These same daf-3 and daf-5 alleles completely block CLD resulting from ts daf-c mutations (Table 2). M37 binding was also observed in dauer-defective daf-6 L2 larvae (Table 4). Because daf-6 mutants have defective head channels leading to the amphid chemosensory neurons (33) and do not respond to dauer pheromone (18), we conjectured that a component other than the dauer pheromone might be inducing ILD. To test whether dauer pheromone activity was sufficient to induce ILD, worms were grown in the presence of an extract further purified from crude extract by silicic acid chromatography. Although capable of efficient dauer larva induction (86-99%), column-purified extract could not induce ILD in wild-type, daf-3(el376), or daf-6(el377) L2 larvae (data not shown). The level of ILD seen in the presence of columnpurified extract (- 10%) and the partial staining observed were similar to that seen in wild-type control worms grown without extract present. This suggests that ILD is not an obligatory part of dauer larva development and that the ILD observed following growth on crude extract is not due to dauer pheromone activity. To eliminate nonspecific antibody binding as an explanation for ILD, srf-2(yj262) L2 larvae that are defective in surface antigen expression (15) were tested for mab M37 binding after growth in the presence of crude extract, and were found to be antigen-negative (Table 4). Moreover, the possibility of artifactual antibody binding to unshed Li cuticles was eliminated by electron microscopy of thin sectioned daf-12(m20) and daf-3(e1376) L2 larvae grown in the presence of crude pheromone extract. No evidence of unshed Li cuticles was found (D. G. Gibson and S.M.P., unpublished results). DISCUSSION The idea that nematodes switch surface composition in response to environmental signals has been based on rapid changes in surface lipophilicity (39, 40) or surface antigenicity (7, 9, 41) that occur during parasitic nematode infections when preparasitic infective larvae enter the definitive host. Here, we show that in C. elegans, display of an Li surface antigen at the L2 stage is inducible by environmental factors (ILD), and constitutive display of this antigen at all larval stages (CLD) results from specific mutations. The genes that result in CLD when mutated include a new gene, srf-6, and six daf-c genes, first studied for their effects on dauer larva formation (19). Interestingly, mutant or wild-type dauer larvae and adults do not bind mab M37, suggesting that different genes may control surface antigen display at these stages. Our mutant screens, performed at 20 C (11), recovered only daf-7 and srf-6 mutants. Perhaps other ts daf-c mutants

12392 Genetics: Grenache et al were not found because at this intermediate temperature they formed dauer larvae, which do not bind mab M37. Besides surface antigen switching and dauer formation, daf-c mutations also affect lifespan (25, 42) and egglaying (43), suggesting that the daf-c gene products are involved in several different processes. Dauer formation occurs in response to environmental signals (17); we investigated whether surface antigen switching was modulated similarly during wild-type development and determined that ILD occurred under altered growth conditions. Both the induction of Li antigen display at the L2 stage and the commitment to non-dauer development (29) occur at the Li molt. Certain ts daf-c mutations result in both CLD and the ts Daf-c phenotype; therefore this temporal correlation may reflect simultaneous action of the daf-c gene products in these two processes. However, our evidence clearly indicates that dauer larva formation and surface antigen switching are controlled differently. The Daf-c phenotype is temperature sensitive, whereas the CLD phenotype of the same mutants is not. This can be explained by the fact that wild-type dauer larva formation is fundamentally a temperature-sensitive process (18). The ts Daf-c phenotype results from alterations in the daf-c genes, whose activities are required to inhibit dauer formation at 25 C, but not at 15-16 C. The mutant daf-c genes are likely to have a loss of function at all temperatures, which at 16 C apparently affects only surface antigen switching. Other evidence that dauer formation and surface antigen switching are controlled differently includes the lack of CLD in daf-2 mutants and the lack of a ts Daf-c phenotype in srf-6 mutants. srf-6- daf-c daf-3/ genesh daf-5 Display of L 1 - antigen in L2-L4 larvae daf-12 df-2 g Dauer larva formation FIG. 3. One of several possible models for the relationship between dauer formation and surface antigen switching based on genetic interactions. Arrowheads indicate positive regulatory interactions (activation), and lines ending in bars indicate negative regulatory interactions (inhibition). Wild-type pathways leading to dauer formation and display of Li antigen in L2-L4 larvae are shown; steps upstream of daf-c genes involving detection of dauer pheromone (e.g., daf-6) and unknown steps involving detection of the putative inducer of L2 larval display (see Results) are not indicated. Dauer-constitutive genes (except for daf-2) are grouped as daf-c and are positioned to indicate their negative effect on dauer formation and antigen expression in later larval stages. The effects on dauer formation are based on interactions between daf-c and daf-d mutations (19). The effects on surface antigen expression are based on interactions described here. Dauer-defective mutations in daf-3 and daf-5 block both antigen expression at stages L2-L4 and dauer formation, whereas daf-12 mutations block only dauer formation. Effects of srf-6 and daf-2 are drawn as separate lines to indicate process-specific effects. The srf-6 gene may also be positioned on the upper branch after daf-3 and daf-s. Proc. Natl. Acad. Sci. USA 93 (1996) The results of testing phenotypes of daf-c;daf-d double mutants fit a model in which the wild-type daf-c gene products (except for daf-2) inhibit both Li antigen display in later larval stages and dauer formation, but through downstream steps controlled differentially by several daf-d genes (Fig. 3). Mutations in daf-3 and daf-5 block both processes; however, daf-12 mutations are dauer-specific, and are therefore likely to act further downstream on a dauer-specific branch (Fig. 3). This scheme is similar to one proposed based on unrelated phenotypic markers; daf-3 and daf-s mutations fully suppress the egg-laying-defective, dark intestine, and clumpy phenotypes of daf-1, daf-7, daf-8, and daf-14 mutations, whereas daf-12(m20) does not suppress these phenotypes (31, 32). Some of our evidence suggests that a parallel pathway may control surface antigen switching independent of the daf genes. The fact that daf-3 and daf-s mutants show partial and variable ILD in response to crude extract indicates that a pathway of induction is functioning in these mutants. In contrast, the CLD resulting from ts daf-c mutations under standard growth conditions is completely blocked by these same daf-3 and daf-5 mutations. Moreover, a daf-3 mutation does not block the CLD resulting from a srf-6 mutation, suggesting that srf-6 may function independent of the daf genes. This interpretation is diagramed in Fig. 3; however, another possibility is that srf-6 acts downstream of daf-3 and daf-5, rather than in a parallel pathway. Although wild-type L2 larvae showed ILD when grown in the presence of crude dauer pheromone extract, the inducer is not the dauer pheromone itself, because dauer pheromone activity is not sufficient for ILD. It appears that an unidentified component present in the crude extract can inhibit surface antigen switching. A daf-6 mutant, which has occluded amphids (33) and does not respond to the dauer pheromone (18), still shows ILD in the presence of crude extract. Therefore, this component must be detected differently than the dauer pheromone. However, it may be detected by the amphids; daf-6 mutants do not respond to the dauer pheromone and other water-soluble substances but apparently can respond to volatile molecules via a distinct set of amphid neurons (44). Whether the signal inducing L2 antigen display is transduced via the daf-c genes, an independent pathway, or both, remains to be determined. Similarities between the products of the ts daf-c genes daf-1 (37) and daf-4 (38) and receptors that mediate cell-cell signaling in vertebrate animals (45) strongly suggest that signal transduction mechanisms may control surface antigen switching. Nematode surface coat molecules are extracellular glycoproteins (6), hence such control could operate on any level, from expression of the apoprotein to post-translational processing steps including glycosylation and localization in the cuticle. Such complications currently prohibit interpretation of CLD or ILD as a change in antigen expression per se. Switching off display of the M37 antigen at the Li molt could represent either a cessation of antigen expression or masking of a persistent larval antigen by other components. The CLD and ILD phenomena could therefore result from unmasking of such a hidden antigen rather than newly induced antigen expression after the Li stage. For srf-6 mutants, certain unmasking phenomena (e.g., removal of a more superficial layer covering an unaltered antigen) are unlikely, because the antigen recognized by the similar mab M38 was not detected in gel immunoblots of total protein extracts of wild-type L4 larvae (11). However, due to technical limitations, these experiments cannot be extended to mab M37 or the daf-c mutants, so that unmasking cannot be ruled out as an explanation for the present results. Structural studies of surface antigens of the parasitic nematode Toxocara canis indicate that they are O-glycosylated (6, 46), a pattern characteristic of vertebrate secreted epithelial mucins (47) and shared by the C. elegans Li surface antigen

Genetics: Grenache et al. surface epitope recognized by mab M38 (11). A mucin-like amino acid sequence is encoded by a cdna for the apoprotein of the major larval T. canis surface protein, TES120 (48). The C. elegans gene let-653 also encodes a mucin-like protein that may be secreted by the secretory/excretory apparatus (49). If nematode surface coat proteins are similar in structure to vertebrate mucins, they may serve a similar function, i.e., to form a protective, yet expendable barrier between the worm and its environment. Artificially manipulating the signals controlling surface antigen switching could prevent a parasite from evading the host immune response. Unlike parasites that must survive hostile host environments, C. elegans is a free-living soil nematode that experiences a consistent environment and apparently would derive no benefit from surface antigen switching. However, nematode species that are congeneric or confamilial with C. elegans are parasites or associates of insects, other arthropods, and snails (50, 51), suggesting that C. elegans may have evolved from parasitic ancestors. In these lifestyles, dauer-like larvae enter the host's body but do not develop. After host death, the nematodes develop and reproduce using the bacterial bloom associated with host carcass decay (52). There may therefore be some adaptive significance to surface antigen switching in insect hosts, which mount immune reactions against foreign invaders (53). We have demonstrated that C. elegans responds to environmental conditions by modifying its surface, a process formally similar to surface antigen switching in parasitic nematodes. 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