Proc. Natl. Acad. Sci. USA Vol. 93, pp. 12388-12393, October 1996 Genetics

Environmental induction and genetic control of surface antigen switching in the 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 can alter their surface coat pro- external conditions, but also might allow formulation of a tein compositions at the molts between developmental stages means to interfere with the immune evasion mechanisms of or in response to environmental changes; such surface alter- highly prevalent, medically important parasites (13, 14). De- ations may enable parasitic nematodes to evade host immune spite its potential significance, the genetic control of surface defenses during the course of infection. Surface antigen antigen expression in nematodes has not been investigated, switching mechanisms are presently unknown. In a genetic except for several preliminary studies identifying C. elegans study of surface antigen switching, we have used a monoclonal surface-altered mutants (10, 11, 15). One of these studies (11) antibody, M37, that recognizes a surface antigen on the first forms the basis for the present work. larval stage of the free-living nematode . We describe here an inducible surface antigen switch in C. We demonstrate that wild-type C. elegans can be induced to elegans, using a monoclonal antibody (mAb) probe that detects display the M37 antigen on a later larval stage by altering the a surface antigen found on Li larvae grown under standard growth conditions. Mutations that result in nonconditional conditions. Under altered growth conditions, L2 larvae are display of this antigen on all four larval stages fall into two induced to display this antigen. We investigated this surface classes. One class defines the new gene srf-6 II. The other antigen switch by isolating (11) and analyzing C. elegans mutations are in previously identified dauer-constitutive mutants that display the Li surface antigen on stages L2 genes involved in transducing environmental signals that through L4 under all growth conditions. Some mutations modulate formation of the dauer larva, a developmentally affect only surface antigen switching, and define at least one arrested dispersal stage. Although surface antigen switching new gene, while others also result in temperature-sensitive, is affected by some of the genes that control dauer formation, constitutive formation of dauer larvae and are in previously these two processes can be blocked separately by specific described genes. mutations or induced separately by environmental factors. The C. elegans dauer larva is a developmentally arrested Based on these results, the mechanisms of nematode surface dispersal stage (16) formed at the second molt, as an alterna- antigen switching can now be investigated directly. tive to the L3, in response to high population density and limited food (17). The genes affecting both dauer larva for- All nematodes have five postembryonic stages (Li through L4 mation and surface antigen switching normally act to prevent and adult) separated by four molts. At each molt a new dauer larva formation in the presence of food, and their multi-layered cuticle is synthesized (1). The internal cuticle activities are required primarily at higher temperatures (e.g., layers, composed primarily of collagens (2, 3), are covered by 25°C) but not at 16°C (18). Hence, loss-of-function mutations an insoluble matrix (2, 4) and the osmiophilic epicuticle (5), in these genes result in a temperature-sensitive, dauer- whose compositions are not well known. Outside of these constitutive (ts Daf-c) phenotype. These genes have been layers is a 5-20-nm-thick surface coat composed primarily of hypothesized to control neural transduction of environmental glycoproteins (for review, see ref. 6). Unlike the other cuticle stimuli during dauer larva formation (19). Their involvement proteins, surface coat molecules are readily secreted into the in surface antigen switching supports the idea that environ- environment (6). mental stimuli also induce surface antigen alterations. How- The molts allow abrupt, wholesale surface changes to occur ever, we also show that surface antigen switching and dauer when a new cuticle is made. Stage-specific surface differences larva formation can be induced separately by specific envi- are known to occur in a variety of nematode species, including ronmental conditions or blocked separately by specific muta- the free-living species Caenorhabditis elegans (6-11). The tions, indicating that the two processes are controlled differ- potential importance of a stage-specific surface composition to ently. is suggested by Trichinella spiralis infections of mammals. Later stages that express different surface antigens escape the immune attack directed against the stages present MATERIALS AND METHODS early in infection (12). Surface composition can also change Nematode Culture for Immunofluorescent Staining. Nem- within a single stage, e.g., in response to a new host or host atode stocks were grown at 16°C on nematode growth agar tissue, and surface molecules can be shed in response to plates seeded w-ith Escherichia coli OP50 (20). For some assays, binding of immune effector cells or antibodies (for reviews, see refs. 6-9). Abbreviations: Li, L2, L3, and L4, first, second, third, and fourth Surface antigen switching results in the restriction of par- larval stages, respectively; mAb, monoclonal antibody; ts, temperature ticular surface molecules to a specific time or developmental sensitive; daf-c, Daf-c, dauer-constitutive; daf-d, Daf-d, dauer- stage. Elucidating the mechanism of surface antigen switching defective; srf, Srf, surface antigen expression; CLD, constitutive larval would not only help explain responses of the nematodes to display; ILD, inducible larval display. tPresent address: Department of Clinical Laboratory Science, Fitch- burg State College, Fitchburg, MA 01420. The publication costs of this article were defrayed in part by page charge §Present address: Department of Molecular and Cellular Biology, payment. This article must therefore be hereby marked "advertisement" in University of Arizona, Tucson, AZ 85721. accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be addressed. 12388 Downloaded by guest on September 24, 2021 Genetics: Grenache et al. Proc. Natl. Acad. Sci. USA 93 (1996) 12389 a few gravid nematodes were placed on plates and allowed to phenotype with daf-1(m40) dpy-13(e184) IV and daf-7(el372) lay eggs for 2-3 h. Parents were then removed and progeny dpy-l(el) unc-32(e189) III. were grown synchronously at the temperatures indicated in Results. Plates. In some studies, nematodes were grown RESULTS on plates containing extracts of depleted culture medium Genetic Analysis of Mutants. When tested on wild-type known to contain dauer-inducing pheromone activity (21). worms grown under standard Conditions, mAb M37 and M38 Crude pheromone extract and a column-purified extract were bound to the surface of Li larvae but not to other stages (11). prepared as described (22). For the latter, pheromone activity In previously described immunofluorescence screens, nine was eluted from a silicic acid column with CHCl3:CH30H (2:1, ethylmethane sulfonate-induced mutants (11) were isolated vol/vol), dried down, redissolved in water, filter-sterilized, and that bind mAb M37 and M38 at stages L1-L4, but not as adults stored at 4°C. (referred to as constitutive larval display, or CLD). Either crude or column-purified pheromone extract was Seven of these mutants have now been analyzed genetically, added to autoclaved nematode growth agar medium (made using mAb M37 surface immunofluorescence as a phenotypic without peptone) just before pouring into 35 x 10-mm Petri marker. Five mutations, including the previously described dishes (2 ml each). The agar was seeded with E. coli OP50 yj43 (11), displayed no distinct phenotype other than CLD suspended in S medium (prepared as described in ref. 23) (e.g., Fig. 1 A and B), showed linkage to unc-4 II and were containing streptomycin sulfate (final concentration, 50 ,ug/ml allelic; these mutations define the srf-6 gene. Three-factor of agar), and used the next day. crosses and deficiency mapping localized srf-6 just to the right Immunofluorescent Staining of Live Nematodes with mAb of dpy-10 II (Fig. 2). M37. Isolation and characterization of mAb M37 were de- Two other mutations,yjll andyjl2, resulted in formation of scribed previously (11). Worms were stained and observed as dauer larvae independent of food limitation when grown at described for mAb M38 (11), except that M37 incubation time 25°C, a phenotype characteristic of ts Daf-c mutants (19). was 2.5 h, and 15 ,ul of fluorescein isothiocyanate-conjugated These mutations showed linkage to dpy-1 III and were allelic secondary antibody was used in a 1.5-h incubation. For pho- with daf-7(el372) III (data not shown). tomicrography, worms were anesthetized with NaN3 as de- CLD in ts Dauer-Constitutive Mutants and srf-6 at Permis- scribed previously (11) and wet mounted on 5% agarose pads. sive Temperature. Based on the ts Daf-c phenotype ofyjll and Strains. Standard C. elegans genetic nomenclature is used yjl2, we tested strains carrying ts daf-c mutations in daf-1, (24). Isolation of strains carrying sff-6(yj5), srf-6(yjl3), srf- daf-2, daf-4, daf-7, daf-8, daf-li, and daf-14 (19) for CLD. 6(yjl5), srf-6(yj43), srf-6(yj41), daf-7(yj11), and daf-7(yj12) has Surprisingly, most of the ts Daf-c mutants showed CLD when been described (11), as has isolation of the sff-2(yj262) mutant (15). Other dauer formation mutants (19), and dauer- constitutive (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 hermaph- rodites. 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 dauer- defective 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 con- structed 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 se- lected 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 ho- mozygous males with unc-4 srf-6(yjl3) II hermaphrodites. FIG. 1. Indirect immunofluorescence of C. elegans mutant strains L2-L4 stage, non-Unc cross progeny were tested for M37 grown at 16°C and stained with mAb M37. Larval stages 1-4, but not immunofluorescence. For complementation testing of srf-6 adults, fluoresce, except in G where the daf-5 mutation suppresses fluorescence at all stages except Li. Images were recorded on TMax with chromosome II deficiencies, srf-6(yjl3) +/srf-6(yjl3) unc- 400 film. (A and B) srf-6(yjl3); (C and D) daf-I(m40); (E and F) 4(e120) males were mated with unc-4(e120) Df/mnCl [dpy- daf-I(m40);daf-12(m20); (G and H) daf-1 (m40); daf-5(el386).A, C, E, 1O(el28) unc-52(e444)] hermaphrodites. L2-L4 Unc-4 prog- and G show epifluorescence images. The same fields are shown as eny containing srf-6(yjl3) opposite the deficiency were bright field images in B, D, F, and H, respectively. (Bar = 0.5 mm.) screened for M37 immunofluorescence. Strains carrying yjli Arrowheads in H indicate the positions of Li larvae that show positive and yjl2 were tested for complementation of their ts Daf-c immunofluorescence in G. Downloaded by guest on September 24, 2021 12390 Genetics: Grenache et aL Proc. Natl. Acad. Sci. USA 93 (1996)

remodeled pharynx, and continued to feed and develop into L4 IIf., larvae and adults at 27.5°C. asmapuzuts 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 srr-,6 constructed double mutants carrying a ts daf-c mutation and a dauer-defective (daf-d) mutation, such as daf-12(m20). The dpy-IO daf-d mutations we used prevent dauer formation even in the /n-26 presence of a ts daf-c mutation (19, 31) and do not themselves Q-L rol-6 unC-4 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). I1 IF In contrast to the daf-12 results, all daf-c mutants carrying a l daf-d mutation in either daf-3 or daf-5 showed M37 binding n IDI 7 --- only at the Li stage (Table 2; e.g., Fig. 1 G and H), suggesting that the wild-type activities ofdaf-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 FIG. 2. Partial genetic map of C. elegans chromosome II, showing the Li molt rather than a defect in antigen expression. A srf-6 the position of srf-6 determined from data presented here. In three- mutation in combination with daf-d mutations exhibited a factor crosses, recombinant progeny ofdpy-10 unc-4/srf-6 parents were third pattern of genetic interaction; neither a daf-12 mutation scored for CLD. Five recombination events occurred between dpy-10 nor a daf-3 mutation blocked CLD (Table 2). In contrast to the and srf-6, and 52 were between srf-6 and unc-4 (data pooled from ts daf-c genes, therefore, srf-6 does not appear to interact with crosses with srf-6 alleles yj43, yjl3, and yjlS). Consistent with these daf-3. results, srf-6(yjl3) failed to complement chromosome II deficiencies CLD of at mnDf3l and mnDf3O, but complemented mnDf6l, mnDf68, and daf-c Mutants Restrictive Temperature. The mnDf88. The position of lin-26 is from ref. 26, positions of other results shown in Tables 1 and 2 were obtained at 16°C, a markers are from ref. 27, and positions of deficiencies are from ref. 28. 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 grown at 16°C (Table 1), a temperature at which a minimal dauer larvae. We refer to all animals that have undergone one fraction of the population is induced to form dauer larvae (17, molt since hatching as L2 larvae, and we do not attempt to 18, 29). With the exception of daf-2 mutants, all were like sif-6 distinguish L2 larvae from the L2d larvae that precede dauer and bound mAb M37 at stages L1-L4, but not as adults (Table formation (17). This distinction is not crucial here, because the 1; e.g., Fig. 1 C and D). Mutants in five different daf-c genes formal definition of CLD requires only that the M37 epitope showed CLD with high penetrance (87-100%), and strains be displayed on larvae that have molted at least once. carrying each of four different daf-li alleles displayed partial mAb M37 binding by L2 and dauer larvae grown under and more variable penetrance (15-76%) of CLD. All three conditions that induced nearly 100% dauer larvae is shown in daf-2 mutants tested appeared similar to wild-type, displaying Table 3. None of the dauer larvae tested displayed the M37 the mAb M37 epitope only on the Li surface, and none of the antigen. However, consistent with the results obtained at 16°C, srf-6 mutants showed a ts Daf-c phenotype at 25°C, the L2 larvae of all daf-c strains tested, with the exception of standard restrictive temperature. Some ts Daf-c phenotypes daf-2(e1370), displayed the M37 antigen. A comparatively are expressed only at 27.5°C (30). Four srf-6 mutants showed small fraction of daf-1l mutant L2 larvae stained. As shown no ts Daf-c phenotype at 27.5°C. In the fifth, srf-6(yj41), 5% of below, M37 antigen display can also be induced in wild-type L2 animals grown from hatching at 27.5°C developed into dauer- larvae. like larvae, but these were incompletely transformed for Environmental Signals Modulate Larval Display of the certain dauer characteristics including a dark intestine and M37 Antigen in Wild Type and Mutants. The decision between dauer and non-dauer development depends on the balance Table 1. mAb M37 immunofluorescence staining of L2-L4 stage Table 2. mAb M37 immunofluorescent staining of L2-L4 double dauer-constitutive mutants grown at 160C mutant larvae grown at 160C Antigen Percent antigen-positive (total no. scored) positive/total daf-d mutant Genotype scored Range (%) allele daf-1(m40) 107/107 100 daf-c or sif daf-12 daf-2(el370) 0/116 0 mutant daf-3 daf-5 m20 m116 daf-2(el369) 1/188 0-1 + 0 (53) 0 (47) 0 (58) 0 (159) daf-2(m41) 1/111 0-1 daf-1 0(107) 0(80) 98 (121) 77 (244) daf-4(m63) 130/132 98-100 daf-4 0 (122) 0 (138) 99 (113) ND daf-7(el 372) 44/44 100 daf-7 0 (124) 0 (97) 99 (146) ND daf-8(el393) 132/136 95-100 daf-8 0 (96) 0 (138) 79 (86) 67 (349) daf-11(m47) 204/347 43-76 daf-lI 0 (103) 0 (94) 24 (104) ND daf-11(m87) 66/134 17-76 daf-14 0 (98) 0 (85) 92 (113) ND daf- 1(sa194) 53/126 15-58 srf-6 95 (136) ND 86 (126) ND daf- 1(sal95) 38/70 24-67 daf-14(m77) 87-100 Results are sums of two experiments on mixed stage stocks, except 153/162 for daf-12(m116), which was tested once. In all cases, Li larvae were Mixed stage nematode stocks were grown and stained as described antigen-positive, and adults were antigen-negative. Mutant alleles in Materials and Methods. Entries combine results of two trials, or four were daf-3(el376), daf-5(el386), daf-11(m47), daf-8(m85), daf- trials for daf- 1(m47). In all cases, Li larvae were antigen-positive, and 4(el364), and srf-6(yjl3); alleles of other daf-c genes were those listed adults were antigen-negative. in Table 1. Downloaded by guest on September 24, 2021 Genetics: Grenache et al. Proc. Natl. Acad. Sci. USA 93 (1996) 12391

Table 3. mAb M37 immunofluorescence staining of daf-c L2 and Wild-type L2 larvae grown in the presence of crude extract dauer larvae grown at 25°C bound mAb M37 (71-99% in four trials, Table 4), indicating Percent antigen-positive that display at a stage later than Li can occur even in wild-type (total no. scored) worms. We refer to this inducible L2 display of the M37 epitope as inducible larval display (ILD). Wild-type dauer Dauer Dauer larvae larvae grown under these conditions did not bind mAb M37. Genotype L2 larvae larvae formed (%) L2 larvae grown on limited food without added extract showed daf-l (m40) 89 (28) 0 (51) 100 a much lower level of ILD (12%, Table 4) and exhibited partial daf-2(el370) 0 (188) 0 (61) 100 or weak staining (not shown). As observed with daf-c; daf-12 daf-4(m63) 97 (38) 0 (61) 98 double mutants (Table 2), three different mutations in daf-12 daf-4(m592) 80 (46) 0 (60) 100 did not interfere with ILD (Table 4). daf-7(e1372) 58 (19) 0 (53) 100 In contrast with daf-12, mutations in dauer-defective genes daf-8(el393) 63 (82) 0 (77) ND daf-3 or daf-5 partially interfered with ILD in L2 larvae grown daf-11(m87) 17 (29) 0 (30) 97 in the presence of crude pheromone extract (Table 4). Al- daf-11(sa195) 3 (33) 0 (19) 100 though the majority of these mutant larvae showed mAb M37 pattern was partial or weak (data Worms were grown on nematode growth plates from eggs laid binding, often the staining during a 3-h period. L2 larvae were harvested for staining after 40-48 not shown). Frequently, only a portion of the body surface h of growth, at which time a few animals on each plate were entering bound M37. Variable staining was obtained even when the the L2-dauer larva molt. Dauer larvae were individually picked from genotype included a daf-1 or daf-7 ts daf-c allele in addition to 3-5-day-old plates and pooled for staining. Dauer formation was a daf-3 dauer-defective mutation. These same daf-3 and daf-5 determined on parallel control plates containing at least 50 animals alleles completely block CLD resulting from ts daf-c mutations per plate. (Table 2). M37 binding was also observed in dauer-defective daf-6 L2 between dauer-inducing pheromone and an antagonistic food larvae (Table 4). Because daf-6 mutants have defective head signal (17). The daf-c genes are believed to represent inter- channels leading to the amphid chemosensory neurons (33) mediate steps in a signaling pathway (19, 31, 32) involving and do not respond to dauer pheromone (18), we conjectured chemosensory neurons that a component other than the dauer pheromone might be processing of environmental cues by inducing ILD. (33-36) and intercellular signal transduction. Indeed, daf-1 To test whether dauer pheromone activity was sufficient to and daf-4 encode putative protein growth factor receptors (37, induce ILD, worms were grown in the presence of an extract 38). The involvement of daf-c genes in surface antigen switch- further purified from crude extract by silicic acid chromatog- ing suggested that surface antigen expression might also be raphy. Although capable of efficient dauer larva induction modulated by environmental signals, perhaps the same ones (86-99%), column-purified extract could not induce ILD in used for dauer formation. To test this, worms were grown from wild-type, daf-3(el376), or daf-6(el377) L2 larvae (data not hatching to the L2 stage on plates containing limited food and shown). The level of ILD seen in the presence of column- an extract of liquid culture medium containing dauer-inducing purified extract (- 10%) and the partial staining observed were pheromone activity (21) and then stained with M37. similar to that seen in wild-type control worms grown without extract present. This suggests that ILD is not an obligatory part Table 4. mAb M37 immunofluorescence of L2 larvae formed in of dauer larva development and that the ILD observed fol- the presence or absence of crude pheromone extract lowing growth on crude extract is not due to dauer pheromone activity. No. of Percent antigen-positive To eliminate nonspecific antibody binding as an explanation Genotype Extract trials (total no. scored) for ILD, srf-2(yj262) L2 larvae that are defective in surface Wild-type dauer + 1 0 (19) antigen expression (15) were tested for mAb M37 binding after Wild-type - 1 12 (94)*t growth in the presence of crude extract, and were found to be Wild-type + 4 92 (495) antigen-negative (Table 4). Moreover, the possibility of arti- daf-12(m20) - 1 16 (55)*t factual antibody binding to unshed Li cuticles was eliminated daf-12(m20) + 3 93 (311) by electron microscopy of thin sectioned daf-12(m20) and daf-12(m1l6) + 3 91 (225) daf-3(e1376) L2 larvae grown in the presence of crude pher- daf-12(m25) + 2 99 (141) omone extract. No evidence of unshed Li cuticles was found daf-3(el376) + 2 87 (259)* (D. G. Gibson and S.M.P., unpublished results). daf-3(mlO) + 1 69 (157)* daf-5(el386) + 3 65 (160)* DISCUSSION daf-7; daf-3 + 1 88 (246)* daf-1; daf-3 + 1 100(130)* The idea that nematodes switch surface composition in re- daf-6(el377) + 1 81 (226) sponse to environmental signals has been based on rapid srf-2(yj262) + 1 0 (195) changes in surface lipophilicity (39, 40) or surface antigenicity All samples were grown for 48 h at 15°C on agar media containing (7, 9, 41) that occur during parasitic nematode infections when no peptone, 50 ,ul of crude pheromone extract (+ extract), or water (- preparasitic infective larvae enter the definitive host. Here, we extract) per 2-ml plate, and 0.5-0.6 mg of streptomycin-treated E. coli show that in C. elegans, display of an Li surface antigen at the OP50 per plate. Alleles not listed were daf-l(m40), daf-3(e1376), and L2 stage is inducible by environmental factors (ILD), and daf-7(e1372). Dauer formation in parallel wild-type + extract plates constitutive display of this antigen at all larval stages (CLD) ranged from 82-99%. results from specific mutations. *Positively staining worms in these trials showed variable results, The genes that result in CLD when mutated include a new including partially and weakly staining individuals. gene, srf-6, and six daf-c genes, first studied for their effects on tWild-type or daf-d L2 larvae were antigen-positive in the absence of pheromone extract only when grown on the modified medium used dauer larva formation (19). Interestingly, mutant or wild-type in this Table; in genetic mapping tests, complementation tests, and dauer larvae and adults do not bind mAb M37, suggesting that the experiments of Tables 1-3, wild-type or daf-d L2-L4 larvae grown different genes may control surface antigen display at these on NGM medium and non-streptomycin-treated bacteria were all stages. Our mutant screens, performed at 20°C (11), recovered antigen-negative. only daf-7 and srf-6 mutants. Perhaps other ts daf-c mutants Downloaded by guest on September 24, 2021 12392 Genetics: Grenache et aL Proc. Natl. Acad. Sci. USA 93 (1996) were not found because at this intermediate temperature they The results of testing phenotypes of daf-c;daf-d double formed dauer larvae, which do not bind mAb M37. mutants fit a model in which the wild-type daf-c gene products Besides surface antigen switching and dauer formation, (except for daf-2) inhibit both Li antigen display in later larval daf-c mutations also affect lifespan (25, 42) and egglaying (43), stages and dauer formation, but through downstream steps suggesting that the daf-c gene products are involved in several controlled differentially by several daf-d genes (Fig. 3). Mu- different processes. Dauer formation occurs in response to tations in daf-3 and daf-5 block both processes; however, daf-12 environmental signals (17); we investigated whether surface mutations are dauer-specific, and are therefore likely to act antigen switching was modulated similarly during wild-type further downstream on a dauer-specific branch (Fig. 3). This development and determined that ILD occurred under altered scheme is similar to one proposed based on unrelated pheno- growth conditions. typic markers; daf-3 and daf-S mutations fully suppress the Both the induction of Li antigen display at the L2 stage and egg-laying-defective, dark intestine, and clumpy phenotypes of the commitment to non-dauer development (29) occur at the daf-1, daf-7, daf-8, and daf-14 mutations, whereas daf-12(m20) Li molt. Certain ts daf-c mutations result does not suppress these phenotypes (31, 32). in both CLD and the Some of our evidence suggests that a parallel pathway may ts Daf-c phenotype; therefore this temporal correlation may control surface antigen switching independent of the daf reflect simultaneous action of the daf-c gene products in these genes. The fact that daf-3 and daf-S mutants show partial and two processes. However, our evidence clearly indicates that variable ILD in response to crude extract indicates that a dauer larva formation and surface antigen switching are pathway of induction is functioning in these mutants. In controlled differently. contrast, the CLD resulting from ts daf-c mutations under The Daf-c phenotype is temperature sensitive, whereas the standard growth conditions is completely blocked by these CLD phenotype of the same mutants is not. This can be same daf-3 and daf-5 mutations. Moreover, a daf-3 mutation explained by the fact that wild-type dauer larva formation is does not block the CLD resulting from a srf-6 mutation, fundamentally a temperature-sensitive process (18). The ts suggesting that srf-6 may function independent of the daf Daf-c phenotype results from alterations in the daf-c genes, genes. This interpretation is diagramed in Fig. 3; however, whose activities are required to inhibit dauer formation at another possibility is that srf-6 acts downstream of daf-3 and 25°C, but not at 15-16°C. The mutant daf-c genes are likely to daf-5, rather than in a parallel pathway. have a loss of function at all temperatures, which at 16°C Although wild-type L2 larvae showed ILD when grown in apparently affects only surface antigen switching. Other evi- the presence of crude dauer pheromone extract, the inducer is dence that dauer formation and surface antigen switching are not the dauer pheromone itself, because dauer pheromone controlled differently includes the lack of CLD in daf-2 activity is not sufficient for ILD. It appears that an unidentified mutants and the lack of a ts Daf-c phenotype in mutants. component present in the crude extract can inhibit surface srf-6 antigen switching. A daf-6 mutant, which has occluded am- phids (33) and does not respond to the dauer pheromone (18), Display of L1 still shows ILD in the presence of crude extract. Therefore, this srf-6- antigen component must be detected differently than the dauer pher- |- omone. However, it may be detected by the amphids; daf-6 in L2-L4 larvae mutants do not respond to the dauer pheromone and other water-soluble substances but apparently can respond to vola- tile molecules via a distinct set of amphid neurons (44). daf-c daf-3/ Whether the signal inducing L2 antigen display is transduced genesH daf-5 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 sig- daf-12 naling in vertebrate animals (45) strongly suggest that signal transduction mechanisms may control surface antigen switch- ing. Nematode surface coat molecules are extracellular glyco- proteins (6), hence such control could operate on any level, from expression of the apoprotein to post-translational pro- Dauer larva cessing steps including glycosylation and localization in the df-2 g formation cuticle. Such complications currently prohibit interpretation of CLD or ILD as a change in antigen expression per se. FIG. 3. One of several possible models for the relationship Switching offdisplay ofthe M37 antigen at the Li molt could between dauer formation and surface antigen switching based on represent either a cessation of antigen expression or masking genetic interactions. Arrowheads indicate positive regulatory inter- of a persistent larval antigen by other components. The CLD actions (activation), and lines ending in bars indicate negative regulatory interactions (inhibition). Wild-type pathways leading to and ILD phenomena could therefore result from unmasking of dauer formation and display of Li antigen in L2-L4 larvae are such a hidden antigen rather than newly induced antigen shown; steps upstream of daf-c genes involving detection of dauer expression after the Li stage. For srf-6 mutants, certain pheromone (e.g., daf-6) and unknown steps involving detection of unmasking phenomena (e.g., removal of a more superficial the putative inducer of L2 larval display (see Results) are not layer covering an unaltered antigen) are unlikely, because the indicated. Dauer-constitutive genes (except for daf-2) are grouped antigen recognized by the similar mAb M38 was not detected as daf-c and are positioned to indicate their negative effect on dauer in gel immunoblots of total protein extracts of wild-type L4 formation and antigen expression in later larval stages. The effects larvae (11). However, due to technical limitations, these on dauer formation are based on interactions between daf-c and experiments cannot be extended to mAb M37 or the daf-c daf-d mutations (19). The effects on surface antigen expression are based on interactions described here. Dauer-defective mutations in mutants, so that unmasking cannot be ruled out as an expla- daf-3 and daf-5 block both antigen expression at stages L2-L4 and nation for the present results. dauer formation, whereas daf-12 mutations block only dauer for- Structural studies of surface antigens of the parasitic nem- mation. Effects of srf-6 and daf-2 are drawn as separate lines to atode Toxocara canis indicate that they are O-glycosylated (6, indicate process-specific effects. The srf-6 gene may also be posi- 46), a pattern characteristic of vertebrate secreted epithelial tioned on the upper branch after daf-3 and daf-S. mucins (47) and shared by the C. elegans Li surface antigen Downloaded by guest on September 24, 2021 Genetics: Grenache et al. Proc. Natl. Acad. Sci. USA 93 (1996) 12393

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