Mutants of Caenorhabditis elegans that form dauer-like larvae

DEVELOPMENTAL

BIOLOGY

126,27&293

(1988)

Mutants of Caenorhabditis elegans That Form Dauer-like Larvae PATRICE Division

of Biological

S. ALBERT AND DONALD L. RIDDLE

Sciences, Tucker Hall, Univmsitg

of Missmwi,

Received August 27, 1987; accepted in revised fm

Columbia, Missmwi

65211

December 9, 1987

The development, ultrastructure, and genetics of two mutants that form dauer-like larvae have been characterized. Dauer larva morphogenesis is initiated regardless of environmental stimuli, and it is incomplete or abnormal. The resistance to detergent characteristic of normal dauer larvae is not fully achieved, and the mutants are unable to exit from the dauer-like state of developmental arrest. Mutant life span is not extended beyond the three weeks characteristic of the nondauer life cycle, whereas normal dauer larvae can live for several months. Growth of &&15(m&)IV, the less dauer-like of the two, is nearly arrested at the second (dauer-specific) molt, but feeding is not completely suppressed. Head shape, cuticle, and intestinal ultrastructure are nondauer, whereas sensory structures (amphid and deirid) and excretory gland morphology are intermediate between that of dauer and nondauer stages. The dclf-9(e1@6)X mutant is dauer-like in head shape, cuticle, and deirid ultrastructure, intermediate in amphid and inner labial neuron morphology, and nondauer or abnormal in the intestine. Also, the dcbf-9mutant exhibits abnormalities in the pharyngeal arcade cell processes and pharyngeal gl gland. Double mutants carrying both d&O and dqff-15 are more resistant to detergent than either single mutant. Like the single mutants, they cannot complete morphogenesis, and they are unable to exit from the dauer-like stage. Both daf-9 and dccf-15 mutations are epistatic to previously described dauer-defective mutations, indicating that these two genes act late in the pathway leading to the dauer larva. The genetic tests and the mutant ultrastructure suggest that the two genes may affect parallel pathways of morphogenesis. 0 1998 Academic Press, Inc.

INTRODUCTION

or tissues exhibiting dauer-specific morphology include the intestine (Popham and Webster, 1979), the excreUnder favorable growth conditions, the postembrytory gland (Nelson et al., 1983), and several anterior onic development of the nematode, Caenwhabditis ele- sensory organs (Albert and Riddle, 1983). gans, consists of four larval stages (Ll-L4) and the The environmental signals influencing both entry adult. In response to overcrowding or scarcity of food into, and exit from, the dauer stage have been described (bacteria) a facultative juvenile stage, the dauer larva, (Golden and Riddle, 1984a). A Caenorhabditis-specific may be formed at the second molt. This specialized dis- pheromone, produced at all stages of the life cycle, appersal form is nonfeeding, arrested in development, and parently serves as a measure of population density especially resistant to environmental stress. Dauer lar- (Golden and Riddle, 1982). The pheromone enhances vae may survive without food four to eight times the dauer larva formation and inhibits recovery. A specific normal 3-week life span (Klass and Hirsh, 1976). When “food-signal” secreted by bacteria acts competitively placed in a fresh environment containing bacteria, with the pheromone to enhance dauer larva recovery dauer larvae begin to feed within 4 hr, resume develop- and to inhibit dauer larva formation. The results of ment, then molt to the L4 stage (Cassada and Russell, behavioral assays suggest that Ll larvae integrate the two competitive chemosensory cues when discriminat1975). Dauer larvae are easily distinguished from other de- ing between alternate developmental fates. Dauer-invelopmental stages. They are relatively thin and dense ducing conditions prolong the L2 intermolt period, and due to radial shrinkage of the body at the dauer-specific result in a morphologically distinct “predauer” L2 molt. About 1 hr after radial shrinkage, dauer larvae larva, called the L2d (Golden and Riddle, 1984a). The acquire resistance to detergent treatment (Swanson LZd larva retains the potential to form either a dauer and Riddle, 1981), presumably as a result of cuticle mod- larva or an L3 larva at the second molt, depending on ification and the occlusion of the buccal cavity by cuticle continuing environmental cues. On the other hand, L2 (Popham and Webster, 1979; Albert and Riddle, 1983). larvae are committed to growth and are unable to form Transverse-section electron micrographs of the body- dauer larvae, even if placed in dauer-inducing condiwall cuticle show a thickened outer cortex and a dauer- tions. specific, striated inner layer (Cassada and Russell, 1975; Our goal in studying dauer larva formation is to unPopham and Webster, 1978; Cox et ab, 1981). Other cells derstand how a set of genes specifies a simple develop0012-1606/88 $3.00 Copyright All rights

0 1988 by Academic Press, Inc. of reproduction in any form reserved.

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Dauw-like

mental sequence. To study the process in mutants, two classes of dauer larva formation (daf) mutant have been characterized. First, temperature-sensitive (ts) dauer-constitutive mutants form dauer larvae at restrictive temperature even in abundant food. When the constitutively formed dauer larvae are shifted to permissive temperature, they exit from the dauer stage and resume development (Swanson and Riddle, 1981). Genetic data suggest that a false, internal signal is responsible for initiation of the dauer developmental sequence (Riddle et ab, 1981; Golden and Riddle, 1985). Second, dauer-defective mutants cannot form dauer larvae and either do not respond to, or do not produce, the dauer-inducing pheromone (Golden and Riddle, 1984b; Golden and Riddle, 1985). Sixteen genes have been ordered with respect to one another in a functional sequence based on the ability of dauer-defective genes to suppress the dauer-constitutive phenotype in certain double mutants (Riddle et ah, 1981). Characterization of sensory abnormalities in several mutants led to the hypothesis that this genetic pathway corresponds to neural processing of environmental stimuli. A subset of dauer-defective mutants (Albert et al, 1981) and one dauer-constitutive mutant (Perkins et al, 1986) have been shown to have ultrastructural defects in sensory dendrites. The present work describes two mutants, daf-15 (m81)IVand daf9(el406)X, which differ from previously described dauer-constitutive mutants in that dauer larva morphogenesis is abnormal or incomplete. Mutant analysis includes electron microscopic examination of selected cells and tissues that normally change morphology upon dauer larva formation. This documents the fact that the mutants are in an intermediate state between the dauer and nondauer. Genetic studies testing the interactions between daf-9 or daf-15 and other daf mutations lead to the conclusion that daf-9 and daf-15 act late in the pathway leading to the dauer larva, and they affect parallel pathways of morphogenesis with differential effects on different tissues. MATERIALS

AND METHODS

Strains and Genetic Nomenclature

Mutant and wild-type (N2) strains of C. &guns var. Bristol were routinely grown on NG agar plates seeded with Escherichia coli strain OP50 (Brenner, 1974). Mutant strains were obtained from Brenner (1974) or the Caenorhabditis Genetics Center. Genetic nomenclature for C. elegans has been described (Horvitz et al,, 1979). Gene names include daf, abnormal dauer formation; dpy, dumpy (short) body; 1012,long body; and uric, uncoordinated movement. Reference stocks for chromo-

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somal rearrangements were MTlOOO, nTl/uncs(e%‘)IV;nTl/dpy-ll(e224)V, and SP309, mnDp33 (X,IV)/+I~unc-2o(ell2)X The nT1 reciprocal translocation suppresses recombination on the right arm of linkage group (LG) IV, including the daf-15 region, and the left arm of LG V. Worms homozygous for nT1 are vulvaless (Ferguson and Horvitz, 1985). The mnDp33 strain contains a duplicated region of the X-chromosome attached to LG IV (Herman et al, 1979). Mutant genes and alleles are listed below according to linkage group: LG I: LG II: LG III: LG IV:

daf-lS(m26), daf-17(m27) daf-22(m130), daf-5(el385) daf-7(el372), daf-2(e1370) daf-18(e1375), dpy-13(e184), uric-24(e138), daf-15(m81), daf-14(m77), dpy-20(e1282), uric-22(m52,s7), uric-26(e205)

LG X: daf-3(e1376),

uric-78(e1217), lon-2(e678), dpy-7(x27), daf-9(e1406), daf-12(m20), daf-20(m25), daf-6(e1377)

Mutant

Isolation

and Mapping

The daf-9(el406)X and daf-15(m8l)IV mutations were induced by ethylmethane sulfonate (EMS) treatment (Brenner, 1974) of wild-type hermaphrodites. Mutants were first identified by visually screening F2 progeny for dauer larvae formed in abundant food. The daf-9 or daf-15 dauer larvae did not recover to reach reproductive maturity. Thus, adult animals from the original plate were “cloned” (allowed to self on separate plates) and screened for segregation of homozygous daf progeny. Heterozygous stocks were then propagated by subcloning and progeny testing, and subsequently backcrossed with dpy-13(el84sd)/+ males to remove unrelated mutations. The semi-dominant dpy-13 marker allows identification of cross-progeny by their semi-dumpy phenotype. Genetic data were obtained by complete progeny counts (Hodgkin et ah, 1979), by complete counts of specific classes of progeny, or by scoring the genotypes of selected recombinants. Map distances or gene orders were determined according to standard methods (Brenner, 1974). Individual animals were handled on the flattened tip of a 0.2 mm diameter platinum wire. Hermaphrodites heterozygous for daf-9 or daf-15 were crossed with dpy-lJ(el84sd)/+ males to test Xlinkage. Semi-dumpy Daf-9 Fl larvae of genotype dpy-13/+;daf9/0 were observed, indicating that &f-9 is X-linked. Cloned hermaphrodites of genotype dpy-13+/+daf-15 segregated F2 Dpy and Daf progeny, but did not segregate the Dpy Daf double mutant, sug-

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gesting that the two mutations are linked. A Dpy segregant that carried a dpy-13 daf-15 recombinant chromosome was mated with wild-type males to generate a semi-Dpy stock of genotype dpy-13 daf-15/++. To order the daf-14 and daf-15 loci on LG IV, hermaphrodites of genotype dpy-13 daf-G++/++daf-14 uric-22 were constructed by crossing dpy-13 daf-15/f+ males with daf-14(m?‘7) uric-.22(m52). The un&?2(m52) allele conveys a dominant twitcher phenotype, so that recombinants could be detected as Wild or Dpy Uric progeny issuing from Uric parents. Of 68 recombinants analyzed, 48 were in the interval between dpy-13 and daf-15 (Dpy Uric phenotype), 4 were in the interval between daf-15 and daf-14 (Wild recombinants that segregated no nonDpy Daf progeny), and 16 were in the interval between daf-1.4 and uric-2.2 (Wild recombinants that segregated Daf-14 progeny). The interval between dpy-13 and uric-22 (6.5 map units) was multiplied by 4/6* (the fraction of all recombinants that occurred between daf-15 and daf-14) to arrive at an estimated 0.4 map units between the two daf genes. The order of auf-9 and lon-2 was verified by determining whether the chromosomal duplication, mnDp33, includes the daf-9(+) locus. An SP309 male stock was generated by crossing SP309 hermaphrodites with N2 males and backcrossing the Wild Fl males with nonUnc SP309 hermaphrodites. Crossing SP309 males with +daf-9/ion-2+ hermaphrodites generated worms of the desired genotype, uric-20+/+daf-9, with and without the duplication. Progeny counts from a worm that had not received the duplication from the heterozygous father were 175 (58%) Wild, 63 (21%) Uric, and 65 (21%) Daf. Combined counts from two populations retaining the duplication were 320 (74%) Wild, 21 (5%) Uric, and 90 (21%) Daf. The fact that uric-20 lies within the duplication is indicated by a reduction in the number of Uric progeny. The percentage of daf-9 progeny, however, was no different from the population without the duplication; approximately one-quarter of the progeny were dauer-like in both cases. Consequently, daf-9(+) was judged not to be included in mnDp33. This is consistent with the placement of daf-9 to the right of Zen-2. Strain Construction

Once genetic mapping permitted the choice of appropriate markers as balancers, two strains were constructed as reference stocks: CB2620, daf-9(e1406)+/ +lon-2(e678)X, and DR412, daf-15(m81)+/+unc24(e138)IV. Additional strains carrying visible markers linked in cis to daf-9 or daf-15 were constructed as follows. Strain DR708, daf-l5(m81)+unc-22(s7)/+dpy-20 (e1282)+IV, was constructed by mating single da$M+/ +unc-24 hermaphrodites with uric-22/+ males. The

vOLUME126,1988

genotype of the maternal parent was verified by scoring the progeny hatched from eggs laid prior to mating. Cross-progeny heterozygous for uric-22 were selected as animals that twitched in 1% aqueous nicotine (Moerman and Baillie, 1979). Homozygous uric-22 (twitcher) adults from a daf-kj+/+unc-22 parent were then selfed animals, and the to identify daf-15 uric-22/+unc-22 daf-15 uric-22 chromosome was balanced over dpy-20 by crossing with dpy-20/+ males. The Fl cross-progeny were tested for segregation of twitching dauer-like larvae and dumpy adults. Strain DR732, daf-l5(m8l)unc-22(s7))/nTlI~+/ nTlV, was constructed using DR708 and MTlOOO,nTl/ Heterozygous daf-15 unc-5(e53)IV;nTl/dpy-ll(e224)V uric-22/++ males issuing from a cross between DR708 and N2 males were selected in 1% aqueous nicotine and mated with MT1000 hermaphrodites. Fl’s that twitched in nicotine were selfed, and subsequently screened for segregation of twitching dauer-like larvae and vulvaless adults. To construct DR733, of genotype +Zon-2(e678)dafWild males (mnDp33/ 9(el406)/unc-78(e1217)+fX, +;unc-78/O) issuing from a mating between SP309 males and uric-78(e1217) hermaphrodites were crossed with km-2 daf-9/h-2+X, a Lon recombinant isolated from the CB2620 reference stock. Wild-type hermaphrodites segregating long, dauer-like larvae and slow, uncoordinated adults in a 1:l ratio (i.e., no longer carrying the duplication) were kept. Developmental

Parameters

Life span. Homozygous daf-15 and daf-9 segregants were handpicked (on Day 0) from 4-day-old stocks of DR412 and CB2620, respectively. In each case, 80 dauer-like larvae were transferred to fresh plates and incubated at 20°C. The number of living, dead, and “missing” larvae was recorded at 2- to 3-day intervals until all the larvae had died (ceased movement and failed to respond to prodding). Worms that could not be found or had died when attempting to crawl up the side of the petri dish were not included when calculating the percentage of survivors. Molting cycle. Development of daf-15 was monitored by scoring pharyngeal pumping (Cassada and Russell, 1975) at 25°C in a synchronous population of daf-15 uric-22 larvae segregated from DR732, daf-15 uric-221 nTlIV;+/nTlVS Eggs were purified by alkaline hypochlorite treatment (Emmons et al., 1979), rinsed, resuspended in 2 ml of M9 buffer (Brenner, 1974), and placed on a shaker at 20°C for 18 hr. Synchronous Ll larvae were collected by centrifugation and placed on one side of a bacteria-free 6 cm agar-filled plate that had a 5 mm wide slice removed from the middle. After the buffer

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Dauer-inducing pheromone was dispensed into the empty dish just before the agar was added. After the agar solidified, 10 ~1 of 5% (w/w) E. coli strain OP50 was added in S medium (Sulston and Brenner, 1974) containing 5 mg/ml streptomycin sulfate. Approximately 12 hr later, the Ll’s were put on the plates and incubated at 25°C in plastic containers to retard loss of moisture. Pharyngeal pumping was monitored as described below on an hourly basis commencing 10 hr after the Ll’s were put in food. L2d larvae from one plate were fixed 7 hr after the end of Ll molt. For morphological comparisons, wild-type dauer larvae from starved cultures were examined in addition to dauer larvae formed in response to the addition of exogenous dauer-inducing pheromone to a well-fed culture. “Starvation-induced” dauer larvae were removed from NG plates 5 days after the supply of bacteria had been depleted. Pheromone-induced dauer larvae were produced from eggs laid at 25°C on agar medium made from autoclaved, clarified S medium obtained from a previous nematode culture (Golden and Riddle, 1982). Nondauers were removed after 2 days; dauer larvae were fixed 2-3 days after the L2d molt. Sample preparation. Most specimens for transmission electron microscopy were fixed with 1% OsOl in 0.1 M sodium cacodylate-HCl, pH 7.3, for 1.5 hr at 28-30°C. However, this fixation protocol did not adequately preserve daf-15 dauer-like larvae. In this case, structural integrity was improved by anesthetizing the worms with 1-phenoxy-2-propanol (3 Ill/ml distilled water) for 2.5 min, rinsing in cacodylate buffer, then fixing in the above 0~0~ solution for 15 hr on ice. The remaining steps apply to all specimens. Buffer-rinsed worms were cut in half and embedded Ultrastructure in agar (Ward et al, 1975). Small agar blocks were deGrowth of nematodes. For electron microscopic analy- hydrated in ethanol and embedded in Spurr’s resin sis, homozygous daf-15 and daf-9 segregants from the (Spurr, 1969). Transverse serial sections approximately reference stocks were handpicked from plates that had 60 nm thick were picked up on unsupported slot grids, been started with a single hermaphrodite and incubated stained with uranyl acetate and lead citrate (Reynolds, at 25°C for 3-4 days. Synchronous wild-type L2 larvae 1963), and placed on lightly carbon-coated Formvar were obtained by one of two methods. Eggs purified by films (Albert et ab, 1981). Sections were photographed alkaline hypochlorite treatment were rinsed, suspended at 40 or 60 kV. in 1.0 ml M9 buffer, and placed on a shaker at 20°C for Larvae prepared for scanning electron microscopy 12-15 hr. The hatched Ll larvae then were collected by were fixed with 3% glutaraldehyde in 0.1 M phosphate centrifugation and put on petri plates with E. coli. Al- buffer, pH 6.8, for 12 hr at 4”C, rinsed with buffer, then ternatively, synchronous L2’s were obtained from eggs water. Larvae were subsequently postfixed in 1% laid by gravid adults during a 2-hr period. Larvae were aqueous OsOl for 7 hr at 4°C rinsed, dehydrated in fixed 2.5-3.0 hr after the Ll-L2 molt, as determined by ethanol, and critical point dried in CO2 (Anderson, observation of pharyngeal pumping. 1951). Specimens were individually mounted on copper Wild-type, pheromone-induced L2d larvae were pre- tape, coated with gold palladium, and photographed at pared by placing -100 synchronous Ll larvae on each 20 kV. of several plates containing pheromone extract (Golden Analysis. Dendrites of major sensory neurons (Ward and Riddle, 1984c). Briefly, each 35 X lo-mm petri dish et al., 1975; Ware et al., 1975; Albert and Riddle, 1983; contained 2 ml of NG agar made without peptone. White et al., 1986) were examined from serial trans-

was absorbed, twitching Ll larvae were transferred individually to the opposite side of the plate. The population of twitchers (daf-15 uric-22) was then transferred in a small volume of M9 buffer to an agar plate with bacteria (at t = 0) and incubated at 25°C. The percentage of worms pumping was determined at a magnification of 100X, using a Wild M5 stereomicroscope. Forty to fifty worms were scored at times up to 18 hr; 45-70 for times between 20 and 37 hr. Development of uric-22 animals was monitored similarly, except that the synchronous Ll’s were placed directly on a plate with bacteria, and approximately 100 worms were scored each time. Length and width measurements. Measurements of daf-9 and daf-15 grown at 20°C were made at 80 and 500X with a Zeiss eyepiece graticule. Worms were placed in 7 ~1 M9 buffer on a 5% agar pad (Sulston and Horvitz, 1977) that contained 3 pi/ml of the anesthetic, 1-phenoxy-2-propanol (Cassada and Russell, 1975). A coverslip was put on the pad temporarily, but removed so worms could be straightened with a flattened platinum wire. Dauer-like daf-9 and daf-15 larvae were picked from plates of CB2620 and DR412, respectively, 4 days after the parental adult had been placed on the plate, and were either measured on Day 4 or transferred to another plate for measurement on Days 7 and 10. Thirteen to sixteen worms were measured each time. Length measurements of daf-15 uric-2.2 and uric-22 individuals grown at 25°C were made as described above at approximately 4-hr intervals between 8 and 36 hr, and at 2, 4, and 7 days. Anesthetic concentrations were 2-4 pi/ml. Six to twelve worms were measured at each time.

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verse-section micrographs through the anterior IO-12 pm of the head. Three duf-15 and four duf-9 dauer-like larvae were analyzed. An additional &f-g larva was sectioned longitudinally. The deirids, excretory gland, and intestine were examined in two to three specimens per mutant strain. Analysis of the deirid neuron was confined to the dendritic tip. Observations of gland morphology centered on the region near which it opens to the excretory duct. The intestine was sectioned in two areas anterior to the gonad. Wild-type structures were examined in the L2, L2d, and’dauer larva stages, two to three worms per stage. In addition, two wild-type dauer larvae were sectioned longitudinally. Gene Interactions Interactions with dauer-defective mutations. Mutants homozygous for either daf-9 or daf-15 and a dauer-defective mutation were constructed to study epistatic relationships (Riddle et al., 1981; Golden and Riddle, 1985). Dauer-defective mutations d&?(e1385), daf-16(m26), daf-17(m27), daf-18(e1375), and daf-22 (ml.%)) were combined with da@ and with a?$16 In addition, daJ=l5 was also tested with the X-linked mutations daf-3(el376), daf-6(el377), daf-12(m20), and dccf-.W(m25).

Construction of strains carrying daf-15 utilized nicotine selection of daf-15 uric-22/++ males issuing from a cross between DR708, daf-15(mSl)+unc-22(s7)/tdpy2O(e1282)+, hermaphrodites and N2 males. The Fl males were crossed with dauer-defective (duf-d) hermaphrodites, and L4 larvae of genotype daf-15 uric-22/ tt;duf-d/+ were selected in nicotine and selfed. Plates were scored for the presence (or absence) of twitching adults in numbers greater than would be expected from the infrequent recombination between daf-15 and uric-22. Adult twitchers segregated from the double heterozygotes were subsequently tested for the recombinational loss of the mutant daf-15 gene. Populations segregating km-2 daf-9;daf-d were scored similarly for the presence of Lon adults. Because duf-9 is X-linked, the heterozygotes were generated by crossing males homozygous for an autosomal daf-d gene with single DR733, tlon-2 daf-9/uric-78++ hermaphrodites. Fl heterozygotes segregating me-78 were discarded. Four plates for each combination of dauer-constitutive, dauer-defective heterozygotes were screened at 20°C for maturation of marked progeny. Interactions with dauer-constitutive mutations. Interactions between either daf-9 or daf-15 and two ts dauerconstitutive mutants, daf%(e1370) or daf-7’(el372), were investigated. The ts dauer-constitutive mutants always form dauer larvae at restrictive temperatures, but grow nearly normally at permissive temperature. Worms ho-

mozygous for either lon-2 daf-9 X or daf-15 uric-22 IV and one of the ts constitutive mutations were obtained as segregants from strains homozygous for the ts constitutive and heterozygous for the non-ts dauer-like constitutive mutation. Strains of genotype daf-2;daf-15 uric-22/t+ and daf-7;daf-15 uric-22/t+ were constructed by crossing nicotine-selected daf-15 uric-22/ males with strains CB1370 (daf-2) and CB1372 tt (duf-7), respectively. The Fl hermaphrodites of genotype daf-2 or daf-7/t;daf-15 uric-22/tt were selected at the L4 stage as twitchers in nicotine, and selfed at 25°C. Dauer progeny, homozygous for the ts constitutive, were shifted to 15°C to resume development. Resultant L4 larvae that twitched in nicotine were selfed at 15°C to verify the presence of daf-15. Homozygous ts dauer-constitutive mutants that were heterozygous for daf-9 were constructed by crossing individual DR733, tlon-2 daf-9/uric-78tt, hermaphrodites with homozygous daf-2 males or daf-7 males. Dauer progeny formed at 25°C from Fl hermaphrodites were placed at 15”C, and recovered L4 larvae were cloned at 15°C to verify the segregation of Eon-2daf-9 individuals. Resistance to detergent treatment was determined by transferring marked, doubly constitutive mutant dauer uric-22 and daf-2 or larvae (daf-2 or daf-7;daf-15 daf-7;lon-2 daf-9) formed at either 15 or 25°C to a drop of sodium dodecyl sulfate (SDS) placed on the lid of an inverted 6 cm petri dish, the bottom of which contained agar to prevent desiccation. Dauer larvae formed at 15°C were SDS-treated 1 week after the parental heterozygote had been cloned; those formed at 25°C were treated after 4 days. The daf-15 double constitutives were treated with 1% SDS for 30 min; the daf-9 doubles with 2% SDS for 50 min. The worms were then transferred to a fresh plate, individually removed from the SDS, and scored as being healthy, sick, or dead. Sick worms were reclassified as dead or alive on the following day after incubation at 15°C and survivors were monitored for the ability to resume normal development. Interaction between daf-9 and dalf-15. Interaction between the two dauer-like mutations was studied at 20°C by examining Lon Uric individuals segregated from a double heterozygote, daf-l5(m8l)unc-22(s7)/ttIV;lon2(e678)daf-9(eleO6)/+ tX, which had been constructed by crossing tlon-2 duf-9/uric-78++ hermaphrodites with duf-15 uric-22/++ males selected in nicotine. Five days after the double heterozygote was cloned, homozygous Lon Uric individuals were scored as dauerlike or adult. The adults were progeny-tested to determine if they had lost a duf allele by recombination. Lon Uric dauer-like larvae exhibited three phenotypes: those with wider, darker bodies (like daf-15), those that were

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thinner and not dark (like d&9), and those that were viduals eventually resume sporadic pumping and unboth thin and dark. Larvae were separated into the dergo some gonadal cell proliferation, but they are three phenotypic groups and treated first with 1% SDS sterile. The daf-15 dauer-like larvae are dark-bodied, as for 12 min (sufficient to kill 92% of daf-15 uric-22) as are normal dauer larvae, but they do not complete radescribed above. Worms remaining on the plates were dial contraction of the body at the second molt. They continue sporadic feeding, and consequently increase treated similarly. After several hours (or the following slightly in size, but they do not reproduce. Neither muday) survivors were treated with 2% SDS for 1 hr (suftant achieves the normal degree of resistance to deterficient to kill 84% of Zen-2daf-9). SDS-treated survivors were put at 20°C to check for the ability to resume gent. Whereas normal dauer larvae survive without harm at least 24 hr in 1% SDS (Albert and Riddle, development. We observed that a high proportion of Lon nonUnc 1983), about 60% of daf-9 dauer-like larvae are killed in worms that looked like daf-9 were resistant to 2% SDS. 2% SDS in 50 min, and daf-I.5 dauer-like larvae are These individuals were placed in 1% aqueous nicotine to sensitive to a 15-min treatment with 1% SDS, as are assay for the presence of uric-22 and presumably daf-15. wild-type L3 larvae. Body size. Body length and width were measured for In a converse experiment, Lon Daf-g-like worms were scored for the presence (or absence) of uric-22, then daf-9 and daf-15 homozygotes segregated from the reftreated with 2% SDS after an overnight recovery pe- erence stocks, CB2620 and DR412, respectively, grown at 20°C. The daf-9 dauer-like larvae were 580 f 20 pm riod. (standard deviation) long and 26 ? 2 pm in diameter (n RESULTS = 55); daf-15 larvae measured 703 f 23 by 32 + 2 pm (n The daf-9 and daf-15 mutants are thus far a unique = 43). By comparison, wild-type dauer larvae formed in mutant type, and they have been characterized to de- response to dauer-inducing pheromone averaged 580 termine the specific aspects of dauer larva morphogen- X 20 pm (Golden and Riddle, 1984a), wild-type L2 larvae esis that are affected by the mutations. Comparisons measured 400 X 25 pm, and L3 larvae (the developmenwere made with the wild-type dauer stage, the predauer tal alternative to the dauer stage) are 590 X 30 pm (L2d) stage, or to other appropriate control specimens. (Cassada and Russell, 1975). The genetic relationships between these two genes and other genes controlling dauer larva development have been determined in order to gain possible insights into the functional sequence, or sequences, represented by the mutations. The results are divided into sections concerning (1) the overall phenotypes, (2) genetic mapping, (3) developmental parameters including growth and life span, (4) mutant ultrastructure including sensory organs, the cuticle and hypodermis, the intestine and specific glands, and (5) gene interactions. A brief summary is presented at the beginning of each section. General Phenotypes Summary. Dauer-like larvae are formed unconditionally by daf-9 and daf-15 mutants, and they never reach reproductive maturity. Strains carrying these mutations were isolated in a visual screen of EMS-mutagenized populations (see Materials and Methods), and stocks must be maintained as heterozygotes. To the untrained eye, the dauer-like larvae are not easily distinguished from normal dauer larvae by observation with the dissecting microscope. Appearance, behavior, and resistance. The daf-9 dauer-like larvae are slender (shrunken radially) as are normal dauer larvae, but have a more transparent appearance than do other dauer-constitutive mutants or pheromone-induced, wild-type dauer larvae. A few indi-

Genetic Mapping

Summary. Genetic crosses demonstrated that the daf-9 and daf-15 mutations behaved as single recessive mutations defining two new genes. Mapping these loci permitted the construction of genetically marked strains to test gene interactions in double daf mutants, as described in the last section. Map positions. The genetic loci defined by the daf-9(el406)X and daf-15(mSl)IV mutations are shown in Fig. 1, along with the positions of reference markers. Two-factor mapping crosses performed are summarized in Table 1, and described under Materials and Methods. Taken together, the genetic data show that daf-9 is approximately 4.4 + 0.6 map units from km-2 and 3.2 + 0.4 map units from dpy-7. It is not included in the duplication, mnDp33. Thus, the daf-9 mutation maps within a cluster of genes close to dpy-6 (Edgley and Riddle, 1987). The order of daf-9 with respect to these genes has not been determined. The daf-15 locus is about 1 map unit from uric-24, between the me-24 and daf-1.4 loci. The neighboring daf-14 locus is defined by a ts dauer-constitutive mutation (Riddle et ah, 1981; Golden and Riddle, 1984b). As determined by complementation testing, daf-14 is within the deficiency mDj7, whereas uric-24 and daf-15 are not (Rogalski and Riddle, 1988). The daf-15 gene is within eDfl8 and eDfl9, but daf-14 lies

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DEVELOPMENTAL BIOLOGY

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TABLE 1 TWO-FACTORCROSSES Genotype of heterozygote l&q-13 +/+ dqf-15 dqf-f-15 uric-26/H uric-2.# doJ15/++ daf-9 bn-2/++ dpy-7 do&g/++

Nonrecombinant phenotypes

Recombinant phenotypes

477 semi-Dpy, 242 Dpy, 230 Daf 229 Uric Daf, 1088 WT 296 Uric Daf, 1230 WT 479 Lon Daf, 1607 WT 634 Dpy Daf, 2452 WT

26 WT 21 Uric, 20 Daf 7 Uric, 7 Daf 46 Lon 49 DPY

Map units WOP) 5.5 3.0 0.9 4.4 3.2

rt 1.0” + 0.6’ It 0.3b f O.6’sc z!z0.4asC

Note. The recombination frequency (p) was calculated from the fraction (R) of recombinant progeny divided by total progeny scored, according to the formulas shown below. Percentage recombination (map units) is given with 95% confidence limits. Daf, dauer-like; Dpy, dumpy; Lon, long; Uric, uncoordinated; WT, wild type. a p = 1 - (1 - 4R)“? bp = 1 - (1 - 2R)“2. ‘Wild-type Daf recombinants were included in the nonrecombinant Daf class.

outside eDfl8 (Hodgkin, 1986). A four-factor cross, utilizing clpy-13 and uric-22 as outside markers, established the gene order, dpy-13. . . daf-15. . . daf-14. . . uric-22 (see Materials and Methods). Developmental

Parameters

Summary. Morphologically, the daf-15 mutant is the less dauer-like of the two, so its growth and molting cycle were examined in detail. This mutant arrested development at the L2 molt as do normal dauer larvae, but sporadic feeding and some increase in body size occurred after the molt. A characteristic trait of dauer larvae is the extension of life span resulting from arrested development and cessation of feeding, so this trait was examined in both mutants. Surprisingly, life span was not extended in either mutant, in spite of arrested growth and greatly reduced feeding.

19

dpy-I3

t eDN9 I

I

z.zz.2;

uric-20

I mnDp33 mnDp33 Ion-2 Ion-2

dpY-20

mDf 7

live for less than 3 weeks (Fig. 2), whereas wild-type dauer larvae are capable of surviving for several months without feeding (Klass and Hirsh, 1976). The mean life span (*standard error) of a daf-15 population was 15.9 * 0.4 days (n = 66); and for a da@ population, 18.9 t 1.2 days (n = 26). Included in the latter value is one worm that lived for 42 days. The mean life spans of the mutants are similar to the 18.5-day mean life span of N2 hermaphrodites at 20°C (Johnson and Wood, 1982). Slightly more than half (43/80) of the daf-9 dauer-like larvae crawled up the sides of the petri dish and died by desiccation in an apparent attempt at dispersal, a behavior characteristic of dauer larvae. Another 14% could not be found. The daf-15 losses were significantly less: 13% died on the sides of plates and 4% were missing, Lost or desiccated individuals were . not included in the data shown in Fig. 2. Molting cycle and growth of daf-15. We examined daf-15 development by monitoring pharyngeal pumping

uric-78 umZ4

eDfl8 1

X

Life span. Dauer-like larvae formed by da@ or daf-15

uric-22

uric-26 dpy-7 dpy-7 u

I mop unit

I i+

dof-9

FIG. 1. Simplified genetic maps of the chromosomal regions including the do&15 IVand da&9 Xloci. Positions of reference markers used

in mapping crosses are shown to the right of the line, and deficiencies and duplications are shown to the left.

0

4

8

12

16

20

24

TIME (DAYS)

FIG. 2. The life spans at 20°C of dqf-9 (open squares) and dqf-15 (closed circles) dauer-like larvae were determined from the percentage of worms alive on a given day.

ALBERTAND RIDDLE

Dauer-like Mutants of C. elegans

(feeding) and body length of dafl5 uric-22 grown in synchronous populations at 25°C. The visibly marked mutant was used because homozygous mutants had to be handpicked from a population of synchronous Ll larvae issuing from heterozygous parents. The Ll larvae homozygous for daf-15 tune-22 were identified by their twitching movement. Pharyngeal pumping is normally suppressed during each of the four molts and in the dauer larva stage (Cassada and Russell, 1975). In comparison with uric-22 controls, the normal growth and feeding pattern of daf-15 uric-22 was interrupted at the second molt (Fig. 3a). Timing of the first two molts was nearly the same. In both strains the Ll molt started 10.5 hr after synchronous Ll larvae were placed on a plate containing bacteria, and the L2 molt began just after 20 hr. Growth and feeding behavior for the strains became dissimilar after this point. By 37.5 hr, uric-22 had completed the L2

277

and L3 molts. During the same interval (20-37 hr), daf-15 uric-22 worms neither completely ceased pharyngeal pumping (as do wild-type individuals destined to become dauer larvae), nor went through any pattern resembling a molt. Instead, the percentage of the population feeding varied from 63 to 93%. After 4 days, 86% (24/28) of the worms were pumping. After 7 days the remaining worms did not appear to be healthy, and none were pumping. Between 8 and 36 hr after being placed in bacteria, the daf-15 uric-22 larvae grew from approximately 290 + 20 pm (average + standard deviation) to 520 f 20 pm; and uric-22 controls grew from 270 ? 20 to 670 + 40 pm (Fig. 3b). Seven-day-old dafl5 we-22 animals were not significantly larger than they were at 36 hr, although uric-22 individuals had doubled in length to adult size (1290 f 50 pm). Ultrastructure

01,

,

1

,

,

,

14

8

a

(

,

,

20

,

,

,

26

TIME

1

,

,

,

38

32

(hrs)

14 I3 1.2

i

2 I.1 2 1.0 1 09F

06-

I2

0.7 -

w 1

0.6 0.4 0.5:

6

b

19

30

41

52

TIME

63

74

85

96

168

(hrs)

FIG. 3. Molting cycles and body length measurements of duf-15 uric-22 (closed circles) and uric-22 controls (open squares) grown at 25’C. Synchronous Ll larvae were placed on NG plates seeded with E. co& at time t = 0. (a) The molting cycles were monitored in synchronous populations by scoring pharyngeal pumping (feeding) at the times indicated. Pumping is suppressed during molts. The daf-15 uric-22 data for the hours between 18 and 36 represent a composite of two experiments. (b) Body length measurements of daf-15 uric-22 (closed circles) and uric-22 controls (open squares). Data represent the average + standard deviation for lo-12 worms measured at each time.

Summary. Features known to exhibit dauer-specific morphology in wild-type animals were examined using electron micrographs of transversely serial-sectioned daf-9 and daf-15 dauer-like larvae. The results of comparisons with wild-type dauer and L2 stages are summarized in Table 2. The objective was to determine whether these ultrastructural features were like the dauer, the nondauer, or intermediate in morphology. The two mutants were found to differ from each other, and different tissues were affected to differing degrees. As indicated in Table 2, both mutants had some sensory structures that were morphologically intermediate between dauer and nondauer. The detailed observations on mutant phenotypes are described below, following the organization of Table 2. During the course of this study, daf-9 was also shown to have defects in the buccal capsule and the dorsal pharyngeal gl gland process. Head morphology. The external features of daf-9 and daf-15 are shown in scanning electron micrographs of the head (Figs. 4a, 4b). The daf-15 dauer-like larva’s anterior tip is similar to that of the wild-type L2 larva, not the dauer larva (Albert and Riddle, 1983). Surrounding the open mouth are six distinct lips, or papillae, which contain sensory dendrites. Inner labial neuron tips (I) are located in the protruding regions closest to the mouth. The anterior tip of the daf-9 dauer-like larva, which is indistinguishable from the wild-type dauer larva, is flatter and covered with a thick layer of cuticle. Although six lips are visible, they are less distinct, and the inner labial sensory structures are not distinguishable. As is the case with wild-type dauer larvae, the anterior portion of the buccal cavity is constricted. Transverse-section transmission electron micrographs show that the daf-9 buccal cavity (Fig. 4d)

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TABLE 2 SUMMARYOF WILD-TYPE AND MUTANT MORPHOLOGY Wild type’ Structure Head morphology Lips Buccal cavity Sensory dendrites Inner labial Amphid AWC: Range of arc Amphid AFD: Finger number Amphid ASG and ASI: Placement Deirids: ADE cilia orientation Cuticle Striated layer Alae Hypodermis Intestine Lumen Opaque cytosomes Excretory gland Secretory granules Cytoplasm (see text)

L2

Mutant* Dauer larva

w9

dqf-15

Distinct Open

Less distinct Occluded

D D

L2 L2

IL2 Anterior -100” 22 + 2 Longitudinal

IL1 Anterior -240” 46 f 2 Posterior Transverse

I I D I D

L2-I -L2 I L2-D

Absent Absent Larger seam cells

Present Present Smaller seam cells

D D D

L2 L2 L2

Open Present

Constricted Present

L2 Absent

L2 Present

Numerous “Active”

Absent “Inactive”

I L2

I L2

a See text for published references. *Most structures were judged to be like those of wild-type dauer larvae (D), second-stage larvae (L2), or intermediate (I) based on criteria described in the text.

is nearly occluded a short distance just posterior to the opening, whereas the daf-15 buccal cavity (Fig. 4~) remains open. Sensory dendrites. There are 60 sensory dendrites in the anterior 80 pm of the hermaphrodite (Ward et al., 1975; Ware et ah, 1975; White et al, 1986). All but six of the processes are grouped into small sensory organs, or sensilla, that are arranged symmetrically around the head. Neurons with dendrites located in channels that penetrate the cuticle are thought to be chemosensory; those with dendrites that terminate within the cuticle are thought to be mechanosensory. Dendrites in each category exhibit dauer-specific characteristics (Albert and Riddle, 1983). Comparisons of mutant dauer-like morphology with wild-type dauer and nondauer features are as follows. Cell names are those used by White et al. (1986). Inner labial neurons. The six inner labial sensilla form a ring around the mouth (Fig. 4). Each sensillum contains two ciliated neurons, designated IL1 and IL2. The dendrites of these neurons differ from one another morphologically and in relative anterior positioning of the sensory tip within the channel. The tip position of IL1 with respect to that of IL2 is altered in the dauer larva, IL1 being more anterior, whereas it is more posterior in the L2 (Albert and Riddle, 1983). In duf-9 dauer-like larvae the inner labial dendritic placement

was intermediate between the L2 and dauer; the distance between the tips of IL1 and IL2 was not significant. The daf-15 sections (three worms) contained an “inner labial-specific” electron opaque precipitate. Consequently, the anterior-posterior order of IL1 and IL2 dendritic tips in this mutant was not determined. Amphidial neurons. The amphid morphology of daf-9 is intermediate between the wild-type L2 and dauer larva, and duf-15 is more like the L2. The amphids are the largest and most structurally complex sensilla. Located in the lateral lips (Fig. 4b), each amphid consists of 12 neurons, eight of which (including ASG and ASI) are exposed to the environment via an open channel. The tips of the other four dendrites (including AWC and AFD) extend into pockets within a support cell that forms part of the channel. The AWC, AFD, ASG, and AS1 dendrites are altered in shape or position in wildtype dauer larvae relative to other stages. The AFD sensory termini are located dorsal to the amphidial channel, and consist of fingerlike projections that extend both anteriorly and posteriorly from a central stalk (Fig. 5). They appear as circles in transverse section (Fig. 6). The number of AFD cell projections is significantly greater in dauer larvae than in both younger and older larval stages (Albert and Riddle, 1983). The average maximum number of projections (*standard error) in wild-type dauer larvae is 46 f 2 (n

ALBERTAND RIDDLE

Dauer-like Mutants of C. elegans

FIG. 4. Scanning and transmission electron micrographs of d&la and daf-9 dauer-like larvae. Morphological features exhibited by da&l5 are characteristic of the wild-type L2, whereas cZ&? resembles the wild-type dauer larva (Albert and Riddle, 1983). (a) Surrounding the open mouth (anterior buccal cavity) in duf-15 are six distinct lips, or papillae, containing the sensory termini of four classes of sensilla (sensory organs). The positions of several sensilla are identified. (bj In d&9 the mouth is nearly occluded, and the head is covered with a thick layer of cuticle that nearly obscures the papillae. (c) Section through da$l5 approximately 1 pm from the anterior tip. Individual sensory neurons are distinguishable. This section is located anterior to the amphids. (d) Comparable region of daf-9 reveals the constricted bueeal cavity. A, amphid sensillum; bc, buccal cavity; C, cephalic sensillum; I, inner labial sensillum; 0, outer labial sensillum. Magnification (a, b) X10,000; (c, d) X14,500.

= 6 neurons), whereas only 22 +-2 (n = 7) are observed in L2 larvae (Fig. 6b). In L4 larvae obtained from recovered dauer larvae the average is 32 + 1 (7~ = 5). The maximum number of AFD projections in do&$ dauerlike larvae (Fig. 6a) was 49 f 2 (n = 8), not significantly different from that of wild-type dauer larvae. The maximum number observed in &f-15 (Fig. 6~) was quite variable, ranging from 12 to 35, with an average of 21 (n = 9). This average is comparable to values observed for the L2 larva (Fig. 6b). However, the cross-sectional

shape of the fingerEke projections in this mutant was often irregular, and they were frequently larger than normal. Neuron AWC extends dorsal and ventral winglike processes from the anterior portion of a centrally located stalk (Fig. 5). In transverse section, these processesform an arc that nearly parallels the nematode’s cuticle (Fig. 7).In wild-type L2 larvae (Fig. ?‘a), the arc seldom spans an area greater than 100”; in wild-type dauer larvae (Fig. 7b) the arc frequently measures 240’.

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DEVELOPMENTAL BIOLOGY

FIG. 5. Side view of amphidial neurons AWC and AFD, reproduced, by permission of the publisher, from Ward et al. (1975). The drawings are based on computer reconstruction of cell outlines from a series of transverse sections through an adult. The dotted circle indicates the position of the basal body or transition zone. Scale bar = 1 pm.

VOLUME 126,1988

In da&15 (Fig. 7~) this dendrite formed an arc nearly the same size or slightly larger than that observed in the L2. The AWC cell arc formed by daf-9 dauer-like larvae is intermediate in range (Fig. 7d). The neuron sheet frequently folded back on itself. This trait is seldom observed in wild-type dauer larvae. In the wild-type dauer larva, the dendrites of neurons ASG and AS1 are displaced posteriorly compared to those in L2 larvae. In both daf-9 and daf-15, the positions of ASG and AS1 within the amphidial channel and sheath cell are intermediate between those observed for L2 and dauer larvae, with the daf-15 neuron position sometimes being more characteristic of L2 larvae (micrographs not shown). Deirid neurons. The tips of the two deirid neurons are located dorsolaterally about 80 pm from the anterior tip in L2 larvae. Each deirid contains a single neuron (ADE), the tip of which is comprised of singlet microtubules aggregated with electron-dense material (Ward et ah, 1975; Ware et al, 1975). In L2 and postdauer L4 larvae these sensory endings are embedded in the body-wall cuticle. A longitudinal view of the dendritic tips is observed in transverse section of the worm. In wild-type dauer larvae, the sensory endings are not embedded in the cuticle, but are held against it by dauer-specific, funnel-like structures associated with the inner cuticular layer (Albert and Riddle, 1983). The

FIG. 6. Amphidial neuron AFD, shown at the region of the maximum number of microvillar-like projections. The projections, which appear as circles in transverse section, are located dorsal to the amphidial channel. (a) The number of daf-9 AFD projections is virtually identical to the number observed in wild-type dauer larvae. (b) Wild-type L2 AFD cell, reproduced, by permission of the publisher, from Albert and Riddle (1983). (c) The number of AFD projections in daf-15 is variable, but approximates that observed in the wild-type L2. Cross-sectional size and shape of the microvillar-like projections (stars) are also variable. AC, amphidial channel; d, amphidial neuron AFD; E, pharyngeal epithelium; m, muscle; tz, transition zone. Magnification (a-c) X27,400.

FIG. ‘7. Micrographs showing the maximum arc formed by the wing-like dendritic tip of amphidial neuron AWC. The exact location of this region may vary among strains and developmental stages. Straight arrows delineate the dorsal and ventral limits of AWC as viewed in transverse section. Arrowheads are used to accent neuron contour. (a) Wild-type L2. (b) Wild-type dauer larva. The star denotes a portion of AWC associated with the opposite (left) amphid. (c) daf-15 resembles nondauer stages. (d) &f-9 has an intermediate arc span and is somewhat abnormal. Part of a region of extensive neural folds, fairly common in this mutant and not in the wild type, is marked by the curved arrow. Dorsal and ventral portions of the arc are not confluent in this section. Magnification (a-d) X15,400. 281

282

DEVELOPMENTAL BIOLOGY

FIG. 8. Morphology of deirid sensory termini. Panels on the left show details of the junctions between the neuron and body-wall cuticle; those on the right show the transverse orientation of the cilium. (a, b) d&I deirids exhibit the cuticular substructure and neuron orientation observed in wild-type dauer larvae (Albert and Riddle, 1983). The electron-opaque sensory tip (a) is embedded in the substructure, a branch of which (arrowhead) extends into the body wall cuticle. (b) The dauer-like morphology and orientation of the &f-9 deirid cilium, approximately 0.5 wrn posterior from the tip. (c, d) c&$-15 deirid morphology can vary within a single individual. Nondauer morphology and orientation are illustrated in c, dauer orienta-

VOLUME 126.1988

ends of the dendrites lie parallel to the longitudinal axis of the nematode, and therefore are cut transversely in transverse section. The deirid tips in daf-9 dauer-like larvae exhibit wild-type dauer structure and orientation (Figs. 8a, 8b). Deirid tips in one daf-15 mutant exhibited both forms of orientation; the left deirid (Fig. 8~) was L2-like and the right deirid (Fig. 8d) was dauer-like. In the other two daf-15 specimens, the deirid tips were cut transversely, as they are in wild-type dauer larvae. The daf-15 deirid tips are embedded in the cuticle as in the nondauer stages. This mutant lacks dauer cuticle morphology, including the subcuticular structure that normally surrounds the dendrite. Cuticle. Internal and external features of the wildtype dauer cuticle distinguish it from that of other developmental stages. In both respects daf-9 larvae exhibit dauer characteristics, whereas the daf-15 cuticle resembles that of the L2. Externally, longitudinal ridges of cuticle (lateral alae) are present for most of the length of wild-type dauer larvae (Cox et ah, 1981). The wild-type dauer ala ridge pattern (Figs. 9c, 9f) is formed by daf-9 (Fig. 9d), whereas daf-15 (Fig. 9e) lacks lateral alae, as does the wild-type L2 (Fig. 9a). Cross-sections of the wild-type dauer larva cuticle reveal a striated basal layer that surrounds the worm, except directly below the lateral alae, where this striated layer narrows and becomes discontinuous (Singh and Sulston, 1978; Popham and Webster, 1978). The dauer cuticle is also thicker than that of the L2 stage. The daf-9 cuticle exhibits dauer traits for these features (Figs. 6a, 8a, 8b); and the daf-15 cuticle (Figs. 6c, 8c, 8d) is more like that of the L2 (Fig. 6b). The slight cuticular thickenings observed near the daf-15 deirid (Figs. 8c, 8d) also appear in the L2 larva (not shown). Hypodermis. The hypodermis, which secretes the cuticle, consists of four longitudinal ridges joined circumferentially by thin sheets of cytoplasm (Sulston et al., 1983). Most of the hypodermis consists of a single syncytium, but there are two longitudinal rows of hypodermal seam cells that are embedded in the lateral hypodermis and give rise to alae in the Ll, dauer, and adult stages. Fluid loss from these cells results in the radial shrinkage of the body that accompanies dauer larva formation (Singh and Sulston, 1978). Transverse sections were made in two areas within the region of hypodermis and intestine located between a ring of four intestinal cells behind the pharyngeal-intestinal valve and the gonad. Morphology was examined

tion in d. The cuticular substructure is absent in this mutant. DC, deirid cilium; Dt, deirid tip; cs, cuticular substructure; sl, striated layer (characteristic of wild-type dauer cuticle). Magnification (a-d) x30,100.

FIG. 9. Wild-type and mutant hypodermal and intestinal cell morphology. Intestinal cells are located dorsal and ventral of a shared lumen. (a) Microvilli in the wild-type L2 lumen are structurally distinct. Intestinal cells contain electron-opaque cytosomes. (b) Intestinal cells of the wild-type L2d, which precedes the dauer larva, are filled with electron-opaque cytosomes; the lumen resembles that of the L2. The cytoplasm of the seam cells is less electron dense than the surrounding hypodermis. (c) Lumen microvilli of a pheromone-induced wild-type dauer larva are 283

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DEVELOPMENTALBIOLOGY

in the mutants, starvation-induced wild-type dauer larvae, and three nonstarved wild-type stages (L2, LZd, and dauer larva). The wild-type L2d and dauer stages were induced by application of exogenous dauer-inducing pheromone to obtain wild-type specimens formed in the presence of abundant food (as were the mutants). Consequently, differences between mutant and wild type observed in the hypodermis or intestine did not result from differences in food supply. Ultrastructure of the lateral hypodermal syncytium, including the seam cells, in the wild type and the dauer-like mutants is shown in Fig. 9. The c&f-9 mutant most closely resembles the wild-type dauer larva, whereas daf-15 resembles the LZd, which precedes the dauer larva. Not all features are clearly evident at the magnifications shown, and some conclusions were made from examination of larger prints. Seam cells in the wild-type L2d stage (Fig. 9b) are larger in transverse section than those of the L2 larva; they are not reduced in size as are the seam cells in wild-type pheromone-induced and starvation-induced dauer larvae, and in daf-9 (Fig. 9d). Seam cell boundaries could not be distinguished in sections of daf-15 (Fig. 9e). The lateral hypodermis of this mutant contains large electron-transparent areas; similar (though smaller) regions are also seen in the wild-t,ype L2d (Fig. 9b). Intestinal cells. The L2 intestine (Fig. 9a) is characterized by a relatively large lumen lined with well-defined microvilli. The intestinal lumens of dauer larvae formed in starved cultures are smaller and the microvilli shorter and less distinct (Fig. 9f). These same features are characteristic of intestinal lumens of dauer larvae formed in response to exogenous pheromone (Fig. 9c). The L2d is a feeding stage, and its intestinal lumen resembles that of the L2. A consistent difference was observed in the relative amount of an electronopaque substance found in the intestinal cells of the various wild-type stages and the mutants. This substance is present in discrete bodies, termed “cytosomes” by Popham and Webster (1979). Organelles within the intestinal cytoplasm are presumably related to energy storage. Some may be lysosomes (Clokey and Jacobson, 1986). Unlike the L2 (or the dauer larva), a large portion of the cross-sectional area of the L2d intestine is occupied by the cytosomes (Fig. 9b). Due to the electron-

VOLUME126,198s

FIG. 10. Ventral (a) and lateral (b) views of the C. elegant excretory gland and neighboring cells reconstructed from transverse serialsection electron micrographs of the L2 larva, reproduced by permission of the publisher, from Nelson et al (1983). The area shown encompasses 30 Frn beginning approximately 65 pm from the tip of the head. Outline of the pharynx and posterior portion of the nerve ring (thin dashed lines) have been included for reference. The origin of the excretory duct is located in the “bridge” joining the two gland processes.E, excretory cell; EC, excretory canal; ED, excretory duct; EN, excretory cell nucleus; EP, excretory pore; G, excretory gland; GN, excretory gland nucleus. Magnification X1830.

dense nature of the matrix, we were unable to determine whether these structures are membrane bound. The intestinal morphologies of the mutants are easily distinguished from one another. The daf-9 mutant (Fig. 9d) does not have lumen traits characteristic of wildtype dauer larvae (Figs. 9c, 9f). The lumen, as in actively feeding stages, is relatively large; microvilli are visible. Although daf-9 intestinal cells contain no electrondense cytosomes, the cells do contain numerous medium-density cytosomes, many of which have a heterogeneous matrix. The intestinal lumen of daf-15 (Fig. 9e) also resembles that of actively feeding stages. This mutant differs from daf-9 in that its intestinal cells contain many electron-dense cytosomes, some of which are quite large. Medium density cytosomes unlike those observed in daf-9 are also present. Thus, with regard to intestinal morphology, daf-15 most resembles the wildtype L2d stage. The daf-9 mutant is abnormal, possessing a dauer-like cuticle and hypodermis, but an intestinal lumen characteristic of feeding stages, and no electron-dense cytosomes.

indistinct; some opaque cytosomes are present. The cuticle has lateral alae. (d) dc&9 intestinal cells possess a morphologically nondauer lumen, but unlike the other stages examined, lack electron-opaque cytosomes (other types are present). The lateral hypodermal seam cells are reduced in size as in normal dauer larvae (cell boundaries indicated), and the cuticle has alae. (e) The morphology of d@15 intestinal cells most resembles that of the L2d. The lateral hypodermis contains large electron-transparent areas, and the cuticle lacks dauer-specific characteristics. (f) Starvation-induced wild-type dauer larva. Intestinal morphology closely resembles that of the pheromone-induced dauer larva, although more numerous vesicles are observed in the hypodermis. H, lateral hypodermis; S, hypodermal seam cell; In, intestinal cell; c, cytosome; L, intestinal lumen; la, lateral ala. Magnification (a) X4800; (b) X4500; (c) X5500; (d, e) X4200; (f) X6500.

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FIG. 11. Excretory gland morphology. The regions shown are located in the cytoplasmic “bridge” between the two gland processes at or near the junction with the excretory duct. (a) In wild-type L2 larvae, this region of the gland is rich in secretory granules. (b) Secretory granules are absent in wild-type dauer larvae (pheromone-induced individual illustrated) and gland cytoplasm is arranged in a loose membranous network. (c) Secretory granules are also absent from &f--15, but the cytoplasmic organization is nondauer. (d) daf-9 gland morphology is similar to that of d&15, but does contain a few secretory granules. EC, excretory cell; ED, excretory duct; G, excretory gland; sg, secretory granules; sm, secretory membrane. Magnification (a, c, d) X28,700; (b) X27,900.

Excretory gland. The gland, part of a four-cell secretory-excretory system (Nelson et ah, 1983), is A-shaped and extends bilaterally symmetrical processes, from cell bodies just behind the terminal bulb of the pharynx, anteriorly to the nerve ring, where the processes join (Fig. 10). The gland processes are also connected by a eytoplasmic “bridge” across the anterior edge of the excretory cell body, where three of the four cells are joined at a junction surrounding the origin of the excretory duct. Sections through this region were used to compare the gland morphology of the wild type with the dauer-like mutants. The bridge between excretory gland processes in wild-type L2 larvae is characterized by a high concentration of secretory granules (Fig. lla). In wild-type dauer larvae, secretory granules are absent, and the gland cytoplasm appears to be arranged in a loose membranous network (Fig. llb). These characteristics

are observed whether the dauer stage is induced by starvation or by exogenously added pheromone in the presence of food (Nelson et al., 1983). The excretory glands of daf-15 and daf-9 (Figs. UC, lid) exhibit traits characteristic of both the L2 and dauer stages. The daf-15 and daf-9 gland cytoplasms are like that of the wild-type L2. However, both mutants are deficient in secretory granules. Granules are absent from the bridge region of daf-15 excretory glands (Fig. llc), and only a few granules are observed in the daf9 glands (Fig. lld). There are no obvious changes in the morphology of the secretory membrane, an organelle that connects the excretory gland with the origin of the excretory duct. Buccal capsule. The daf-9 dauer-like larvae exhibit ultrastructural defects in tissues associated with the buccal capsule, which functions in the initial uptake of food from the environment. The dauer larva buccal

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capsule is occluded by the cuticle (Fig. 12). The cheilostom (K) is enclosed by the lips and is lined with body wall cuticle. Prostom (P) cuticle is underlain by arcade tissue, mesostom (M) cuticle by pharyngeal epithelial cells, and the metastom (M’) and telostom (T) by muscle cells. In wild-type adults (Wright and Thomson, 1981) and dauer larvae (Fig. 12) the pharyngeal cuticle and underlying tissues are closely apposed. However, in daf-9 dauer-like larvae these structural components are separated by an interstitial matrix that may contain invaginations of arcade or epithelial cell membrane (Figs. 13a-13c). The apparent consequence of the loose association of pharyngeal cuticle with the arcade cells is loss of its characteristic transverse triangular shape (Figs. 13b, 7d). In five animals examined, the degree to which the cuticle is misshapen was found to be variable, and generally more severe in the prostom (Fig. 13b) than mesostom (Fig. 13~). The presence of epithelial cells (and coincident hemidesmosomes and tonofilaments) seems to act as a stabilizing force in normal animals (Wright and Thomson, 1981). A second abnormality observed in daf-9 is specific to the arcade cells, which form a ring of tissue around the most anterior portion of pharyngeal cuticle. The posterior processes of these cells contain electron-dense granules (Fig. 13~). In the wild type, the presence of granules in arcade cells (and epithelium) is associated with molting (Wright and Thomson, 1981), but such granules are smaller and more numerous than those observed in daf-9. gl gland. Morphology of this gland in Pharyngeal wild-type adults has been described by Albertson and Thomson (1976). Dorsal and subventral gl cell bodies are in the terminal bulb of the pharynx. The dorsal gl cell sends forward a process that opens through a cuticle-lined duct into the pharyngeal lumen of the telostom. (Subventral processes open into the lumen at the back of the metacarpus, the anterior pharyngeal bulb.) Micrographs of the anterior regions of the dorsal gl processes in wild-type dauer larvae and daf-9 dauer-like larvae are shown as viewed in longitudinal (sagittal) section through the point where the duct enters the

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pharyngeal lumen (Figs. 12a, 13a), and in transverse section about 1.5 pm posterior to the duct opening (Figs. 12d, 13d). Although this process is enlarged near the duct opening in both strains, the daf-9 process is also enlarged posteriorly. Transverse sections through daf--15 dauer-like larvae also indicate some degree of enlargement relative to the wild type (not shown). Gene Interactions

One way to examine a possible functional relationship between two genes affecting a developmental sequence is to determine the phenotypes of double mutants. Such mutants might exhibit only one of the two phenotypes (genetic epistasis), both mutations might be expressed, or a new phenotype might be exhibited. This section concerns the interactions between daf-9 or daf-15 and either dauer-defective or ts dauerconstitutive mutations. Two of the characteristics that distinguish daf-9 and daf-15 dauer-like larvae from normal dauer larvae (the degree of resistance to detergent and the ability to resume development when put on a fresh grid of bacteria) were examined in the doubly constitutive mutants and in daf-9; da&l5 double mutants. Taken together, the genetic tests place both daf-9 and daf-15 at the end of the genetic pathway for dauer larva formation, but the gene functions were not ordered with respect to each other. Interactions with dauer-defective mutations. In contrast to previous results obtained with dauer-constitutive mutants that complete dauer larva morphogenesis (Riddle et al., 1981), none of the dauer-defective mutations tested were capable of suppressing expression of daf-9 or daf-15. Worms heterozygous for either daf-9 or daf-15 and a mutation in any one of nine dauer-defective genes were constructed as described under Materials and Methods. The chromosome carrying the dauer-like constitutive gene also carried a linked visible marker (i.e., Eon-2 daf-9 or daf-15 me-22). The only marked adults segregated from animals heterozygous for daf-9 or daf-15 and an unlinked dauer-defective mutation were ion-2 or uric-22 recombinants that had lost the Summary.

FIGS. 12 AND 13. Buccal capsule and pharyngeal gl gland of wild-type dauer larvae (Fig. 12) and daf-9 dauer-like larvae (Fig. 13). Approximate locations of transverse-section insets are indicated by dashed lines to the longitudinal section. In the wild type, pheromone-induced dauer larva (lea), arcade and epithelial tissue (underlain by the prostom and mesostom, respectively) lie in close apposition to the pharyngeal cuticle. The dorsal process of the gl gland is relatively small, although some swelling is evident adjacent to the duct opening (arrow). In duf-9 (13a), an interstitial matrix separates the arcade and much of the epithelial tissue from the pharyngeal cuticle, and the gl dorsal process is swollen. An arrow denotes the duct opening. Transverse sections through the prostom cuticle show that the buccal cavity of the wild-type dauer larva (12b) is triangular compared to that of a d&g specimen (13b). Transverse sections through the metastom show the wild-type structure (12~) and a more normal looking dqf-f-9 (13c), but invaginated epithelial cell membrane is found in the interstitial areas (arrowheads). Arcade cell processes in dd9 contain electron-opaque granules; such structures are uncommon in wild-type dauer larvae. Comparison of transverse sections through the dorsal process of the gl gland in wild-type (12d) and doJ-9 (13d) shows that the gland is enlarged in daJ9. a, arcade tissue; ag, arcade granule; bc, buccal cavity; E, pharyngeal epithelium; gl, pharyngeal gl gland; K, cheilostom; M, mesostom; M’, metastom; mx, interstitial matrix; Nu, nucleolus; P, prostom; T, telostom. Magnification (12a, 13a) X11,000; (12b, 13b) X22,100; (12c, 13~) X16,900, (12d, 13d) X11,700.

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FIG. 13

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15°C (permissive temperature for daf-2 and daf-7) were more resistant than either parent. Resistance of daf-9 itself to SDS also exhibited temperature sensitivity. A greater fraction of km-2 daf-9 worms grown at 25°C were resistant to SDS (38%) than their 15°C counterparts (6%). This result was not expected because the formation of daf-9 dauer-like larvae is not temperature-dependent. Double constitutive, dauer-like larvae formed at 25°C also were more resistant than those formed at 15°C (with the exception of daf-7; Zen-2 daf-9 dauer-like larvae, at least 95% of which were resistant at both temperatures). Double constitutive dauer-like larvae that survived SDS treatment were transferred to plates seeded with bacteria and monitored for resumption of development at 15°C. Nearly 100% of the dauer larvae formed by daf-2 and daf-7 at 25°C will begin feeding and develop into adults within l-2 days. The only putative doubleconstitutives that resumed development within a 2week period were subsequently shown to be genetic recombinants, and not homozygous for the dauer-like constitutive gene. Interactions between daf-9 and daf-15. Genetically marked d&15; daf-9 worms were more resistant to SDS than either mutant parent. They were, however, unable to exit the dauer-like state and resume development at 20°C. Worms homozygous for both daf-9 and daf-15 were picked as Lon Uric segregants from daf-15 uric-ZZ/++IV;Zon-2 daf-S/++X at 20°C. There were three phenotypic classes among the Lon Uncs, referred to as LU-9 (body thin and clear, like daf-9), LU-15 (nonshrunken dark body, like daf-15), and LU-9-15 (thin, dark body). No adult Lon Uric worms were observed. Handpicked Lon Uric worms were grouped according to phenotype and treated with 1% SDS for 12 min to

linked daf mutation. We conclude that the tested dauer-defective mutations are not epistatic to (cannot mask the presence of) the daf-9 or daf-15 mutations. These results place dclf-9 and daf-15 after daf-3, daf-5, and da$E in the genetic pathway (Fig. 14). In other words, expression of the dauer-like phenotype is not suppressed by any of the mutations that block the dauer formation pathway, so the steps defined by daf-9 and dafl5 must formally be placed after those blocks. Interactions with ts dauer-constitutive mutations. A series of crosses was performed to determine whether a ts dauer-constitutive mutant that forms structurally normal dauer larvae can mask the nonconditional dauer-like phenotype of daf-9 or daf-15. Temperaturesensitive dauer-constitutive mutations representing both branches of the dauer formation pathway (Fig. 14) were chosen for these tests, which were performed at both 15 and 25°C (Table 3). The daf-7(el%%s) mutant hyperresponds to the dauer-inducing pheromone, whereas daf-Z(elb70ts) does not (Golden and Riddle, 1984b). In the gene combinations tested, daf-9 and daf-15 were epistatic to the ts dauer-constitutive mutations with respect to overall appearance and the inability to recover from the dauer-like state. However, resistance to SDS was affected by both genotype and growth temperature. Double mutants with daf-2 or daf-7 generally were more resistant to SDS treatment than daf-9 or daf-15 alone, and the daf-7’ multiples generally were more resistant than those formed with daf-2. Less than 50% of the double constitutives formed with daf-15 were resistant to SDS. The double constitutives formed with daf-9 were more resistant to SDS treatment than doubles formed with daf-15. This latter difference is consistent with the more dauer-like morphology of the daf-9 parent. In addition, the daf-9 doubles formed at

ENVIRONMENTAL CUES da/ -22 i

&PHEROMONE i

SENSORY

dot-18 I daf-8 Adof-7-& Adof-,, I i

dof-IO

I

I

k-of-2 i

PROCESSING

daf-I? I

daf-6 I daf-I4

I

I ! i

289

Mutants of C. elegans

daf- 3 daf- 5 daf-12 I

daf-lb daf-20 I

Ldaf-I&

i

MORPHOGENESIS

daf-4

I

I I

-&+

-

I

daf- 15

DAUER -LARVA

da/-9)

I

OTHER 4

-i

i

FIG. 14. A genetic pathway for dauer larva formation, one of several possible representations, based on epistatic relationships (Riddle et aL, 1981; Golden and Riddle, 1985; this work). Mutations represented are epistatic to those positioned on their left. Gene names of dauer-constitutives are given in the pathway itself to represent points where false signals may be initiated. Mutations in dauer-defective genes block the pathway at the positions shown by dashed lines. Genes shown in bold face are those that have been tested in this work for epistatic relationships with d&9 and a?$15 For genes other than d&9 and daJ-15, all possible constitutive-defective combinations have been constructed.

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DEVELOPMENTALBIOLOGYVOLUME126.1988 TABLE 3 SDS RESISTANCEOFDOUBLECONSTITUTIVEMUTANTS

FORMEDATTWOTEMPERATIJRES Percentage SDS resistant” Genotype

15°C

25°C

c&f-15 uric-22 do&?;dqf-15 uric-22 w-7; w-15 uric-22 km-2 daj-9 do&2; h-2 do&f-9 do&Y; h-2 do& dqf-2

0 (O/125) 0 (O/147) 19 (7/37) 6 (101168) 74 (1051142) 95 (73/77) 0 (O/-900) 10C

0 (O/160) 31 (33/106) 41 (591143) 38* (102/2’72) 100 @l/81) 100 (64164) 100 (SO/SO)

W-7

100 (93193)

Note. Larvae of the above genotypes were transferred to a drop of SDS on the lid of an inverted, agar-filled petri dish. After treatment, all worms were transferred to a plate seeded with bacteria, and those alive the day after treatment were scored as resistant. “da&l-15 multiple mutants were treated with 1% SDS for 30 min; dclf-9 multiples, with 2% for 50 min. *In three replicate treatments, SDS resistance of km-2 duf-9 individuals varied from 19 to 52%. This amount of variability was not observed at 15°C or for other mutant combinations. ‘Result from Swanson and Riddle (1981).

determine whether the double mutant was more resistant to SDS than duf-15 uric-2.2 alone. Populations remaining on the plates were also treated with SDS. None (O/32) of the handpicked LU-15 worms survived detergent treatment, and none were obtained from treatment of total populations. All of the handpicked LU-9 (8/8) worms survived. About 50% (7/15) of the handpicked LU-9-15’s survived, and 13 additional worms were picked up in the total population screen. Thus, 20 LU-9-15 worms survived the first concentration of SDS and could later be tested for resistance after 1 hr in 2% SDS. Twelve (60%) survived. This exceeded the percentage of survival exhibited at 20°C by the auf-9 lon-2 control (28/180 = 16%) and the c&f-15 uric-22 control (5/60 = 8%). Many of the worms that survived 1% SDS treatment of the total populations were Lon nonUnc and looked like duf9. Eighty-eight percent (53/60) of these also survived the 1-hr treatment in 2% SDS. This raised the possibility that increased resistance might result from being heterozygous for duf-15 me-22. To test this hypothesis, Lon nonUnc Daf-g-like individuals were picked as Fl’s from the marked heterozygote and placed in 1% aqueous nicotine. Sixty-six percent (38/58) twitched in nicotine, and thus were duf-15 uric-22/++IV;lm-2 duf-9 X The nontwitchers, ion-2 da&?, numbered 20 (34%). These numbers are consistent with the expected Mendelian ratios. After an overnight incubation to recover from nicotine treatment, 64% (21/23) of the worms het-

erozygous for daf-15 uric-22 survived a 1-hr treatment in 2% SDS, whereas only 11% (2/B) of the nontwitcher group survived. It is possible that the two surviving nontwitchers carried recombinant duf-l/i+ chromosomes. We conclude that the duf-15 me-22/++ background of Eon-2 daf-9 increases resistance to SDS. DISCUSSION

Virtually all tissues of the nematode change morphology during development of the dauer larva. The cuticle acquires a unique ultrastructure at the L2d-dauer molt, and becomes detergent resistant. The intestinal lumen contracts and the content of storage granules within the intestinal cell cytoplasm is transformed. The hypodermis preferentially shrinks to reduce the diameter of the dauer body and increase its density. Glandular ultrastructure differs markedly, and both mechanosensory and chemosensory organs assume a dauer-specific morphology. These dauer-specific changes are reversed upon exit from the dauer stage. The arrangement and ultrastructure of cells in same-stage isogenic animals are largely invariant (Ward et al., 1975; Ware et al., 1975; Lewis and Hodgkin, 1977; Albert et ab, 1981; Chalfie and Thomson, 1982; White et al., 1986; Perkins et al., 1986). In addition, little variation from adult neuron structure and arrangement is observed in growing (nondauer) larval stages (Albert and Riddle, 1983). Consequently, comparisons between relatively small numbers of individuals (two to four) are valid. A limitation on the analysis of mutant phenotypes is that some differences from the wild type might be due to closely linked, secondary mutations unrelated to the one that is scored genetically. Although our duf-9 and daf-15 mutants were generated under standard conditions for EMS mutagenesis, and our stocks have been extensively backcrossed to unmutagenized genetic backgrounds, the unlikely possibility that the daf-9 or duf-15 stocks might harbor more than one mutation cannot be eliminated. Nevertheless, both mutations segregate as single loci in genetic mapping crosses, and the phenotypic analysis reported here was performed only after maintenance of the mutations in heterozygous stocks for many generations. Two general classes of daf mutant have been described previously (Riddle et al., 1981). They are dauerdefective mutants, which are unable to form dauer larvae, and dauer-eonstitutive mutants, which form morphologically normal dauer larvae in the presence of abundant food. The dauer-defective class consists of three types: mutants that do not respond to the dauerinducing pheromone, one mutant that does not produce the pheromone, and mutants that exhibit a normal behavioral response, but form SDS-sensitive dauer larvae. The latter class is abnormal in morphogenesis. The

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The d&9 and daf-15 mutations are epistatic to all dauer-constitutive class also consists of three mutant types. First, there are ts mutants that hyperrespond to dauer-defective mutations tested. This genetic result the dauer-inducing pheromone at permissive tempera- places both daf-9 and daf-15 at the end of the genetic ture, and form dauer larvae even in the absence of pathway as it is currently known. Our interpretation of pheromone at restrictive temperature (Golden and the genetic and phenotypic data is that daf-9 and daf-15 Riddle, 1985). Null (amber) alleles of two of these genes, act beyond the point at which the normal physiological daf-4 and daf-7, exhibit a ts dauer-constitutive pheno- switch into the dauer is activated. Instead, these mutations result in incorrect expression of only a portion of type, presumably reflecting the wild-type temperaturedependence for both entry into, and exit from, the dauer the dauer larva morphogenetic sequence. Certain mularva stage (Golden and Riddle, 1984b). Mutant dauer tations in genes for hormone receptors on specific tarlarvae formed at restrictive temperature are induced to get tissues might be expected to have such a phenotype. mutations affecting exit from the dauer stage when shifted to permissive By contrast, dauer-constitutive earlier steps in the genetic pathway result in a global temperature. Mutant adults are abnormal in egg-laying behavior (Trent et ab, 1983). Second, mutants of one switch to dauer larva morphogenesis, and in the case of ts mutants, a behavioral response to a temperature gene, daf-2, do not hyperrespond to the dauer-inducing pheromone, they are not defective in egg laying, and downshift permits recovery from the dauer stage. No order of function could be genetically determined they differ in their epistatic relationships to dauer-defor daf-9 and daf-15 with respect to each other. The fective mutations. The daf-9 and daf-15 dauer-like mutants described effects of these two genes were somewhat additive in here represent a third class of dauer-constitutive mu- daf-9;daf-15 double mutants. Resistance to SDS was somewhat enhanced, even in duJQ/daf-9;daf-15/+ anitant that is nonconditional in expression and abnormal in dauer larva morphogenesis. These mutants are un- mals. However, these mutants did not form morphologable to exit from the dauer-like state. The daf-9 dauer- ically normal dauer larvae, and they were unable to exit like larvae shrink radially, and acquire the dauer head from the dauer-like state. We interpret this to mean morphology and cuticle ultrastructure. Some of the that other functions, acting in parallel with the daf-9 daf-9 sensory structures are dauer-like and some appear and daf-15 pathways, are needed to complete dauer to be intermediate between dauer and nondauer mor- larva morphogenesis (see Fig. 14). The daf-9;daf-15 douphology. The daf-15 dauer-like larvae acquire LBd-like ble mutants were variable in phenotype, as though the intestinal morphology, and are intermediate in some daf-9 and daf-15 phenotypes were partially exclusive. sensory structures, but they are not dauer-like in head Based on their appearance in the dissecting microscope, shape or cuticle ultrastructure. only about one-third of the doubly mutant dauer-like The functional significance of the dauer-specific spe- larvae assumed a mixed daf-9 plus daf-15 morphology. cializations is largely a matter of speculation. However, The majority had the appearance of either daf-9 or it seems likely that the occluded mouth and multidaf-15 alone. Perhaps there is some variability in the layered cuticle both contribute to the resistance to envi- timing with which daf-9 and daf-15 dauer-like pathways ronmental insult, as measured by SDS resistance. In- are executed. Progress along one of the abnormal pathtestinal differences presumably reflect the feeding vs ways prior to initiation of the second might result in an nonfeeding state, and changes in chemosensory den- overall predominance of the former body morphology. drites may account for differences between dauer and On the other hand, nearly simultaneous execution of the nondauer chemotaxis (Albert and Riddle, 1983). The two pathways might result in the mixed phenotype. daf--15 deirid (mechanosensory) morphology suggests The mutant phenotypes suggest that daf-15 might be that changes in the alignment of the ADE cilium in the arrested earlier in dauer larva development than daf-9, dauer stage are not simply a secondary effect of which is the more dauer-like of the two mutants. The changed cuticle structure. Five out of six daf-15 ADE pattern of pharyngeal pumping observed in daf-15 larcell tips examined were oriented transversely as in the vae was unlike that of normally developing worms, or dauer, but the cuticle was like that of the L2, and it worms entering the dauer stage. In wild-type individcompletely lacked the funnel-like cuticular substrucuals destined to become dauer larvae, pharyngeal ture that is normally associated with the deirid in the pumping stops upon entry into the second molt, and dauer stage. Hence, the assumption of dauer-like neuro- remains suppressed until environmental conditions imnal orientation seems to be independent of the dauer prove (Cassada and Russell, 1975). This is also true for cuticle morphogenesis. In the normal dauer larva, the mutants that form dauer larvae constitutively (Swancuticular substructure and the orientation of the ADE son and Riddle, 1981). By contrast, daf-15 individuals cilium may both affect the sensitivity or function of this entering the second molt did not completely cease phasensillum. ryngeal pumping. This suggests that daf-15 dauer-like

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larvae may be more like L2d (predauer) larvae than dauer larvae. The nondauer-like head morphology and the L2d-like intestinal morphology of daf-1.5are consistent with this view. Several observations lead to the conclusion that da,9 and dafl5 result in irreversible abnormalities in morphogenesis. If morphogenesis were simply incomplete, then the ts dauer-constitutive mutations that result in formation of normal (complete) dauer larvae at restrictive temperature should be completely epistatic to daf-9 and daf-15. This is not the case. The ultrastructural study of daf-9 dauer-like larvae revealed some abnormal tissues. Defects in the pharyngeal cuticle and arcade cells were correlated with loss of the normal triangular shape of the buccal cavity. Arcade cells contained abnormal granules, and the pharyngeal gl gland was enlarged. Albertson and Thomson (1976) suggested that the gl gland might secrete digestive enzymes, or possibly aid in degradation of the old cuticle during molting. Darkly staining granules move forward through the dorsal process just prior to ecdysis (Singh and Sulston, 1978). This type of granule was not seen in daf-9 or wild-type dauer larvae. The pharyngeal and intestinal abnormalities in daf-9 suggest that this mutant could be deficient in the uptake of nutrients, in spite of the fact that daf-9 Ll and L2 larvae seem to pump normally. Genetic and behavioral studies show that food limitation by itself is not sufficient to induce formation of dauer larvae, but that high population density (high levels of dauer-inducing pheromone) is normally required. Nutritionally limited (starving and crowded) daf22 animals that do not produce the pheromone do not form dauer larvae, or dauer-like larvae (Golden and Riddle, 1985). Hence, it seems unlikely that the pharyngeal and intestinal abnormalities in daf-9 cause the dauer-like syndrome. Instead, they seem to be pleiotropic effects of the da39 mutation not clearly related to the intermediate state between dauer and nondauer. Analysis of daf-9 and daf-15 show that the dauer vs nondauer switch is not absolute, but that intermediate states are possible if morphogenesis is perturbed genetically. The animals appear to be in an intermediate state because development is interrupted at the second molt, and some structures are dauer-like whereas some are not. In one daf-15 animal, the bilaterally symmetrical deirids differed from each other, one being dauerlike and the other nondauer. Certain cells, particularly the excretory gland and amphidial neurons, were found to be intermediate in morphology in both mutants. This implies that the differentiated state of cells in the dauer larva may result from a global, quantitatively variable signal such as that created by variation in hormone concentrations. In the daf-9 and daf-15 mutants, the

VOLUME126,1988

hormonal balance may not be completely shifted to fully achieve the dauer state, so that target tissues exhibit intermediate responses. Even in daf-15, the least dauer-like of the two mutants, growth is slowed at the second molt and soon stops entirely, in spite of the fact that sporadic feeding continues (see Fig. 3). This suggests that an early, or “low threshold,” aspect of dauer larva morphogenesis may be developmental arrest, and that normal dauer larvae are arrested in development not simply as a consequence of a lack of feeding, but as a consequence of other genetic and physiological controls. It is intriguing that in both daf-9 and daf-15, life span is not extended as it is in normal dauer larvae (Klass and Hirsh, 1976), in spite of the fact that sexual maturation is prevented, and feeding is at least partially suppressed. The separation of increased life span from arrest in growth and development suggests that the extension of life span requires a portion of the normal dauer larva program that is not activated in either of the dauer-like mutants. This work was supported by DHHS Grant HD11239 and Research Career Development Award HD00367 to D.L.R. We thank Dr. Merton Brown and David Pinkerton of the Experiment Station Electron Microscope Facility for drying, coating, and photographing specimens for scanning electron microscopy. We also thank Dr. Samuel Ward for allowing us to reproduce his illustration of amphidial neurons, and Dr. Teresa Rogalski for helping select d&15 uric-22 Ll larvae. REFERENCES ALBERT,P. S., BROWN,S. J., and RIDDLE, D. L. (1981). Sensory control of dauer larva formation in Caenorhabditis elegans. J. Cmp. NG-wo~. 198,435-4X

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