Temporal regulation of cuticle synthesis during development of Caenorhabditis elegans

DEVELOPMENTAL

84.2’7’7-285

BIOLOGY

(1981)

Temporal Regulation of Cuticle Synthesis during Development of Caenorhabditis elegans GEORGE N. Cox, MEREDITH Division

of Natuml

Sciences,

Thimann Received

KUSCH, KARIN DENEVI,

Laboratories, June

26, 1980;

University accepted

of California, in revised

AND ROBERT S. EDGAR Santa

Cruz,

Santa

Cruz,

California

95064

form December 8, 1980

The pattern of cuticle protein synthesis during development of the nematode Caenorhabditis elegans has been studied using NaHX4C0,. Both pulse-labeling and pulse-chase-labeling experiments indicate that synthesis of cuticle components occurs at high levels during the molting periods and at much reduced rates during the intermolt periods. No such discontinuous pattern is observed for the synthesis of total noncuticle macromolecules during development. The soluble and insoluble proteins of the cuticle, which comprise the inner and outer cuticle layers, respectively, follow similar patterns of synthesis during the two molts examined. At each molt the structural components of the cuticle account for approximately 10% of the total macromolecules labeled by NaHWO,. No evidence is found for reuse of cuticle material between successive developmental stages of C. elegans.

INTRODUCTION

These proteins are extremely insoluble due to nonreducible covalent cross-links. Cuticle formation in the small soil nematode, CaenorAt present, very little is known of the temporal prohabditis elegans, is a potentially favorable model system gram of nematode cuticle synthesis during development. for the study of a complex assembly process in a multicelMicroscopy studies on a variety of nematode species lular organism. The cuticle is a multilayered extracelluhave shown that the hypodermis undergoes dramatic lar structure elaborated by an underlying layer of hypo- morphological changes at each molt associated with the dermal tissue, which, for the most part, consists of a secretion of the new cuticle (Kan and Davey, 1968; Johnsingle large syneytium that extends throughout the son et al., 1970; Singh and Sulston, 1978). At these times length of the animal (White, 1974). During the animal’s 3 the thickness of the hypodermis increases up to twofold, day development the cuticle is replaced at each of four the hypodermal cytoplasm becomes swollen with large Golgi bodies and numerous ribosomes, and there is an inpostembryonic molts. Although considerable differences of RNA within the hypodermal are found in the architecture of the cuticle between dif- creased accumulation ferent nematode species and often between different de- cells. These observations suggest that the hypodermis velopmental stages of the same species (summarized in becomes activated for the synthesis of proteins, presumBird, 1971), all nematode cuticles appear to be similar in ably cuticle proteins, in large quantities at each molt. their basic biochemical composition. The typical nema- This notion is supported by the work of Leushner and tode cuticle consists largely of protein with small quantiPasternak (1975) who measured total collagen synthesis ties of lipid and carbohydrate usually also present (Bird, during postembryonic development of P. silusiae and ob1971). The inner cuticle layers are composed primarily of served bursts of synthesis preceding each ecdysis. Since collagen-like proteins that are covalently linked to each the cuticle is largely collagenous, Leushner and Pasterother through disulfide bonds (McBride and Harrington, nak proposed that these increases were mainly due to increased cuticle collagen synthesis at these times. With 1967; Leushner et al., 1979; Cox, Kusch, and Edgar, manuscript submitted). Three genetically distinct colla- the development of methods for the purification of intact cuticles from C. eleguns (Cox, Kusch, and Edgar, manugen chains have been detected in the cuticle of adult Ascaris lumbricoides (Evans et al., 1976) while a larger script submitted) it became possible to address this quesnumber appear to be present in the cuticles of adult Pan- tion directly. In this report we describe the pattern of cuagrellus silusiae (Leushner et al., 1979) and adult Caen- ticle protein synthesis during the final two molts in the orhabditis elegans (Cox, Kusch, and Edgar, manuscript life cycle of C. elegans. In accord with the proposal of Leushner and Pasternak (1975) we find that the synthesubmitted). The outer layers of the cuticle are composed of proteins that are chemically distinct from the collagens sis of cuticle proteins by C. elegans is modulated during and have been named “cuticlins” (Fujimoto and Kanaya, development in concert with the molting cycles. In addition, we find that there is little if any reutilization of cuti1973; Cox, Kusch, and Edgar, manuscript submitted). 0012-1606/81/080277-09$02.00/O Copyright All rights

0 1981 by Academic Press, Inc. of reproduction in any form reserved.

DEVELOPMENTALBIOLOGY

278 cle material

between different

developmental

stages of

C. elegans. MATERIALS

AND

METHODS

Nematode strains and culture. The general methods of nematode culture used were those described by Brenner (1974). Nematodes were grown on 100 mm NG or 8P agar plates (Brenner, 1974; Schachat et al., 1978) using E. coli strain OP50 as a food source. The Caenorhabditis elegans temperature-sensitive sterile mutant, ferl(hclts)I, was used throughout most of these studies. At the restrictive temperature of 25”C, mutant hermaphrodites lay only unfertilized oocytes due to a defect in sperm development (Ward and Miwa, 1978). Dauer larvae were obtained by mass growth on egg white plates and purified from other developmental stages by incubation with 1% sodium dodecyl sulfate (SDS) and centrifugation through 15% Ficoll as described by Cox, Kusch, and Edgar (manuscript submitted). Stock solutions of dauer larvae were stored at 16°C in M9 buffer (Brenner, 1974) until use (usually within 2 weeks). Pulse-labeling experiments. Labeling experiments were usually performed in duplicate with independent groups of worms and each set of experiments was repeated at least twice. The dauer larvae for each time point (usually 5-10 x 103) were added to individual agar plates in 0.5 ml of M9 buffer and incubated at 25°C to permit development. At the appropriate times nematodes were rinsed from these plates with M9 buffer, washed three times by low-speed centrifugation (2 min at 200g) to remove contaminating bacteria, and incubated for 60 min with gentle agitation in a capped tube containing 0.5 ml of M9 buffer and 15 PCi and NaH14C0, (New England Nuclear, 40-60 mCi/mmole; l-2 mCi/ml). At the completion of the labeling period, free label was removed from the worms by washing quickly four times with 10 ml of chase media (667 ml M9 buffer, 8.4 g NaHCO, to 1 liter). The worm pellets were frozen in a dry ice-acetone bath and stored at -20°C until extraction. The number of worms in each tube was estimated by removing three 100~~1 aliquots prior to the final centrifugation and counting the worms in each aliquot visually with the aid of a dissecting microscope. Pulse-chase labeling experiments. Growth of worms and labeling procedures were as described for the pulselabeling experiments except that the labeling periods were reduced to 30 min, the amount of label increased to 60 &i (L4 cuticle experiments) or 30 &i (adult cuticle experiments), and the worms washed for 60 min with a total of 10 changes of chase media at the completion of the labeling periods. Following these washings, nematodes were placed onto fresh agar plates and allowed to develop to the appropriate ages at 25°C. Room tempera-

VOLUME 84, 1981

ture averaged 20°C during these experiments. Prior to sonication, nematodes were rinsed from these plates, washed free of bacteria, counted, and chilled on ice for 30 min. Cuticle isolation procedures. Cuticles were purified by sonication and incubation with 1% SDS essentially as described (Cox, Kusch, and Edgar, manuscript submitted). Cuticles remain insoluble under such conditions due to extensive cross-linking between the component proteins. Nematodes were sonicated (10 X 0.3 min) on ice in 2.5 ml of sonication buffer [lo mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride (PMSF)] and the cuticle pieces collected by centrifugation for 4 min at 300g. Aliquots (800 ~1) of the supernatants were mixed with 250 pg carrier bovine serum albumin, made 10% with trichloroacetic acid (TCA), and allowed to sit on ice for at least 60 min. TCA-insoluble material was collected by centrifugation and TCA-soluble radioactivity determined by scintillation counting of the supernatants. The TCA-insoluble pellets were washed twice with ice-cold 10% TCA, once with ethanol: ether (1: 1) or acetone, and solubilized by heating overnight at 37°C with 0.5 ml of Protosol (New England Nuclear) prior to scintillation counting. Residual cellular material was removed from cuticles by incubation of the crude cuticle pellets in 0.5 ml of ST buffer [l% SDS, 0.125 M Tris-HCl (pH 6.8)], at 100°C for 2 min and agitating gently overnight at room temperature. Initial experiments showed that nearly all SDS-soluble radioactivity was present in macromolecules so for most experiments TCA-insoluble and SDS-soluble radioactivity measurements were combined to yield a value for total noncuticle macromolecules. SDS-soluble radioactivity generally represented 10% or less of this total. In some experiments radioactivity in the cuticle pellets was determined at this point by solubilization with Protosol as described above, while in the other experiments the cuticle pellets were further extracted by heating for 2 min at 100°C in 0.5 ml of ST buffer, 5% p-mercaptoethanol (BME) to release the disulfide cross-linked proteins of the cuticle (referred to as soluble cuticle proteins). The BME-insoluble cuticle material (insoluble cuticle proteins) was washed quickly three more times with ST buffer, 5% BME at room temperature, and solubilized with Protosol before counting. All radioactivity measurements were obtained by counting loo-p1 samples in 10 ml of 3a70B liquid scintillation cocktail (Research Products International) in a Beckman LS-250 liquid scintillatin counter. Enzyme digestion studies. Labeled nematodes were sonticated in 2.5 ml of Tris-HCl (pH 7.4) and crude cuticle material collected by centrifugation. Aliquots (100 ,ul) of the supernatants were mixed with 700 ,ul of digestion buffer [0.145 M NaCl, 10 mM Tris-HCl (pH 7.4)], con-

COX ET AL.

C. elegans Cuticle Synthesis

taining 100 pg of either pronase (Sigma, Type VI protease), ribonuclease (Sigma, Type IIIA), or deoxyribonuclease (Sigma, Type I) and incubated for 4 hr at 37°C. DNase digestion buffer also contained 5 mM MgSO, and 1 mM PMSF, the latter to inhibit any serine proteases (Price et aZ., 1969). Reactions were stopped by the addition of ice-cold TCA to 10% and TCA-insoluble radioactivity measurements determined as described above. Control samples were processed in parallel incubations minus enzyme. Amino acid analyses. Samples for amino acid analysis were hydrolyzed in 6 N HCl for 4 hr at 145°C (Roach and Gehrke, 1970) and the analyses performed on a Beckman 121MB amino acid analyzer. Effluents (30-100 ~1) from the column were collected using a fraction collector and assayed for radioactivity. The recovery of 14C counts applied to the analyzer was always 100%. Lethargus measurements. Pharyngeal pumping was used as an index of lethargus (Cassada and Russell, 1975). Fifty nematodes were observed with the aid of a dissecting microscope for each time point and animals showing no movement of the posterior bulb of the pharynx after several seconds were considered not pumping. Length measurements. A Wild M5 dissecting microscope equipped with an eyepiece graticule was used to measure the lengths of individual worms. Nematodes were straightened by mounting on glass microscope slides and passing briefly several times through the flame of a bunsen burner. This procedure does not significantly alter nematode length. RESULTS

Dauer Larua Recovery and Growth The life cycle of C. elegans normally consists of four juvenile stages (designated Ll, L2, L3, and L4) and the adult. Molts serve to separate one developmental stage from the next and are named for the stages they terminate. Each ecdysis is preceded by a period of lethargus during which pharyngeal pumping (feeding) and nematode locomotion are suppressed. An individual nematode typically spends 2 hr in each lethargus and the old cuticle is shed shortly after the recommencement of pharyngeal pumping (Singh and Sulston, 1978). Lethargus periods allow the molting cycles to be followed and the degree of synchrony of populations quantitated. In response to adverse environmental conditions such as starvation, L2 juveniles will molt into a specialized resistant stage called the dauer larva, which is a developmental alternative to the normal L3 juvenile. Dauer larvae do not feed or grow and can survive for many times the normal life span without significant effect on subsequent development (Cassada and Russell, 1975; Klass and Hirsh, 1976). When placed on fresh medium dauer larvae resume feed-

279

ing and reenter the molting cycles. Dauer larvae molt twice during subsequent development, the hrst molt transforming them into L4’s and the second into adults. The morphological events of these molts and the molts in the regular life cycle appear identical (Sing and Sulston, 1978). As dauer larvae develop to adults they increase in length by more than twofold, from 500 to 1300 pm (Cassada and Russell, 1975). This growth occurs in a continuous manner not limited to the molts and is typical of growth during the normal life cycle. Because dauer larvae can be easily isolated in large numbers, they provide a convenient means for obtaining large synchronous populations of L4’s and adults for biochemical studies of cuticle synthesis during development. NufP4C03

Characterization

The synthesis of cuticle proteins during development of dauer larvae to adults was studied using NaH14C0,. NaH14C0, presumably enters the worm as 14C0, which diffuses freely through the cuticle. Incorporation of Na14C0, into TCA-insoluble macromolecules is very rapid (within 10 min) and is dependent upon the animals being alive. Under our incubation conditions a maximum of l-2% of the label is incorporated into TCA-insoluble macromolecules during a 60-min pulse. This value is comparable to values we have obtained using a variety of radioactive amino acids (our unpublished results), although it is less than that usually obtained when worms are pulsed with 35S-labeled E. coli (Garcea et al., 1978). The incorporation of NaH14C03 into macromolecules by C. elegans is relatively specific for amino acids in proteins. Enzyme digestion studies (data not shown) indicate that 65-80% of the radioactivity incorporated into TCA-insoluble macromolecules during a 60-min pulse can be converted to a TCA-soluble form by treatment with pronase. Neither DNase nor RNase has any significant effect. To determine which amino acids become labeled by NaH14C0,, soluble macromolecules and purified cuticles (obtained from adult animals that had been incubated with NaH14C03 for 40 min during the L4 molt) were acid hydrolyzed and the amino acids separated on an amino acid analyzer. Effluents from the analyzer were subsequently monitored for radioactivity and positions of 14C radioactivity peaks compared to the elution profiles of 3H-marker amino acids. At least 65% of the radioactivity present in soluble macromolecules and at least 75% of the radioactivity present in purified cuticles could be accounted for as labeled amino acids in these experiments. The remaining radioactivity eluted from the analyzer as one major and several minor peaks between glutamate and proline and its identity is not known. It is possible that this material represents small peptides resulting from incomplete hydrolysis of our samples. No radioac-

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VOLUME 84, 1981

Cuticle Synthesis during

0

’ -1 0 Label,“g

1 4

I 8

/ 12

, 16

Permd Chase

f Hours)

FIG. 1. NaHW03 chase characterization. A large synchronous nematode population was incubated with NaHW09 for 30 min, washed with chase media for 60 min, and divided among several fresh agar plates to continue development at 25°C. At the times indicated, nematodes from individual plates were harvested and TCA-soluble (W-W) and TCAinsoluble (A -A) radioactivity measurements determined. Data represent the means plus the range of values for two experiments performed in parallel.

tivity eluted from the analyzer at positions corresponding to amino sugars. Alanine, aspartate (asparagine), glutamate (glutamine), and glycine are the amino acids that become significantly labeled after treatment of C. elegans with NaH14C0,. These amino acids become labeled in both cuticle and noncuticle proteins. Glutamate (glutamine) is the most heavily labeled amino acid, accounting for at least 40% of the amino acids labeled in both cuticle and noncuticle proteins. Some minor and variable incorporation of NaH14C0, into arginine, leucine, proline, and serine was also observed in some experiments. Figure 1 demonstrates that unincorporated NaH14C0, canbe effectively “chased” from the animals following a short pulse. A population of nematodes was incubated with NaH14C0, for 30 min, washed with chase media for 60 min, and portions of the population assayed for TCAsoluble and TCA-insoluble radioactivity at various times during subsequent development. In experiments of this type, less than a 10% change in the amount of TCA-insoluble radioactivity is usually observed between 1 and 16 hr of chase. In contrast, TCA-soluble radioactivity decreases rapidly during the first l-2 hr of chase and by 4 hr approaches a plateau level which characteristically represents one-fifth to one-tenth of the TCA-insoluble radioactivity level. The amount of this residual TCA-soluble radioactivity is not appreciably decreased with longer chase periods.

Development

I. Pulse-labeling experiments. The pattern of incorporation of NaH14C0, into cuticle and noncuticle macromolecules during development of dauer larvae to adults is shown in Fig. 2. At the various times indicated, synchronous nematode populations were given 69-min pulses of NaH14C0,, extracted to separate cuticle and noncuticle macromolecules (see Materials and Methods) and the amount of radioactivity in each fraction determined.fer-l(M), a temperature-sensitive fertilizationdefective strain of C. elegans, was used for these studies to avoid possible ambiguities that might have arisen as a consequence of eggshell synthesis [fertilized eggs secrete chitinous shells that copurify with cuticles (Cox, Kusch, and Edgar, manuscript submitted)] or of cuticle synthesis by young juveniles developing in utero. Greater than 90% of the macromolecules labeled by NaH14C0, at all times during development are noncuticle macromolecules. Incorporation of NaH14C0, into noncutitle macromolecules increases contintuously with age (Fig. 2) and in a manner that is fairly proportional to nematode length (data not shown). The rate of increase is greatest during the L4 stage when the growth rate is also at its maximum. No deviations from this pattern are observed during the lethargus periods when the nematodes are not feeding. In contrast to the results observed for noncuticle macromolecules, incorporation of NaH14C0, into cuticle material during development follows an oscillating pattern,

Recovery

Time

(Hours)

FIG. 2. Incorporation of NaHWO, into cuticle and noncuticle macromolecules during development of dauer larvae to adults at 25°C. Synchronous nematode populations were grown from dauer larvae to the times indicated, incubated with NaHWO, for 60 min, washed four times with chase media, and frozen immediately. After thawing, radioactivity in cuticle (0-O) and noncuticle (A-A) macromolecules was determined. Pharyngeal pumping (A-A) was monitored in parallel during development. Data represent the means plus the range of values for two experiments performed in parallel.

COX ET AL.

C. elegans Cuticle Synthesis

281

I

Rscovery

Time

(Hours)

H’w*dt

A*covery

Tim*

I Hours

)

FIG. 3. Synthesis of L4 (a) and adult (b) cuticle and noncuticle macromolecules during development from dauer at 25°C. Several synchronous nematode populations were grown simultaneously from dauer larvae and at the times indicated different sets of nematodes were incubated with NaHW08 for 30 min, washed with chase media for 60 min, and placed back onto fresh agar plates to continue development. When these populations had grown for a total of 15.5 hr from dauer for the L4 cuticle experiments or 27 hr from dauer for the adult cuticle experiments, they were harvested and the amount of radioactivity in noncuticle macromolecules (A-A) and soluble (0-e) and insoluble (O-O) cuticle components for each group determined. Pharyngeal pumping (A-A) was monitored in parallel during development. Radioactivity data represent the means plus the range of values for two experiments performed in parallel.

with two sharp peaks of activity occurring 11 and 19 hr after dauer larvae are placed onto agar plates (Fig. 2). The timing of these peaks coincides with the lethargus peaks of the dauer and L4 molts, respectively. During the intermolt periods and after the fmal molt into the adult, incorporation of NaH14C0, into cuticle material is much lower than at the lethargus peaks but still above background. Approximately two to three times as much radioactivity is incorporated into cuticle material during the L4 lethargus as during the dauer lethargus. This result is consistent with the approximate twofold difference in size of the animals at these times (Cassada and Russell, 19’75). 2. Pulse -chase experiments. The oscillating pattern of incorporation of NaH14C0, into cuticle material observed in the pulse-labeling studies is consistent with the notion that cuticle protein synthesis is a discontinuous process. However, our isolation methods measure only secreted and cross-linked cuticle material; therefore, other explanations are possible for these results. For example, cuticle protein synthesis could occur at a constant rate during development but one or more cellular elements necessary for the secretion and/or cross-linking of these proteins might exhibit a molt-dependent pattern of activity, resulting in an increased rate of cuticle deposition at these times. To distinguish between these possibilities, pulse-chase experiments were used to determine directly the times of synthesis of the structural components of the L4 and adult cuticles. For these experiments synchronous nematode populations were grown from dauer larvae to the various times indicated in Fig. 3, different sets of worms incubated with NaH14C0, for 30 min, washed with chase media for 60

min, and placed onto fresh agar plates to continue development. When these nematode populations had developed for a total of 15.2 hr from dauer for the L4 cuticle experiments or 27 hr from dauer for the adult cuticle experiments, they were harvested, and the amount of radioactivity in cuticle and noncuticle macromolecules for each group determined. The synthetic patterns of the soluble and insoluble portions of the L4 and adult cuticles were followed separately in these experiments by treating purified cuticles with a sulfhydryl reducing agent (see Materials and Methods). These two classes of cuticle components comprise the inner and outer layers of the cuticle, respectively. The results obtained for the L4 cuticle will be considered first (Fig. 3a). Synthesis of L4 cuticle components is fist detected 6-8 hr after dauer larvae are placed in contact with a fresh food source. This is approximately l-2 hr before the onset of the dauer lethargus. As the population enters the dauer lethargus a rapid increase in the synthesis of both soluble and insoluble L4 cuticle components is observed. The synthesis of these two groups of cuticles components obtains a maximum at the lethargus peak and then declines in parallel with the resumption of pharyngeal pumping, signaling the completion of the dauer molt. The synthetic ratios of these two groups of cuticle components vary slightly at different times during this molt, with the ratio of insoluble to soluble cuticle synthesis being greatest during the early phases of the molt and decreasing thereafter. Overall, approximately 60% of the total cuticle material synthesized during this molt is of the soluble class. Synthesis of L4 noncuticle macromolecules, in contrast, increases continuously with age. No decline is ob-

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FIG. 4. Time course of appearance and disappearance of label into L4 cuticle and noncuticle macromolecules during development. A large synchronous nematode population was incubated with NaHW03 for 30 min during the middle of the dauer molt, washed with chase media for 60 min, and divided among several fresh agar plates to continue development at 25°C. At the times indicated, nematodes from individual plates were harvested and radioactivity in cuticle (0-O) and noncutitle (A-A) macromolecules determined. Pharyngeal pumping (A-A) was monitored in parallel during development. Radioactivity data represent the means plus the range of values for two experiments performed in parallel.

served following recovery from the dauer molt but rather a continued increase. At the lethargus peak of this molt the structural components of the L4 cuticle account for 7-9% of the total macromolecules labeled by NaH14C0 For thladult nematode (Fig. 3b) we observed a pattern of synthesis and accumulation of cuticle and noncuticle macromolecules that was qualitatively similar to that found for the L4 juvenile. Adult cuticle synthesis is first observed 14-16 hr after dauer larvae are placed onto plates. This is a few hours before the onset of the L4 lethargus. Synthesis of both soluble and insoluble adult cuticle components increases rapidly as the population enters the L4 lethargus, reaches a maximum at the lethargus peak, and then decreases as the population completes the molt. Synthesis of adult noncuticle macromolecules does not display this pattern, but rather increases steadily during development. During this molt, a fairly constant ratio of about 2: 1 is maintained for the synthesis of soluble to insoluble cuticle components. The structural components of the adult cuticle account for lo-15% of the total macromolecules labeled by NaH14C0, at the lethargus peak of this molt. 3. Kinetics of appearance and disappearance of label into L4 cuticle material. The failure to detect any signifi-

cant incorporation of NaH14C0, into adult cuticle components until just prior to the L4 lethargus in the above experiments suggests that L4 cuticle material is not reused by the nematode to form the adult cuticle. To corroborate this finding, we followed the fate of labeled L4 cuticle material over time. A large synchronous nematode

population was given a 30-min pulse of NaH14C0, during the middle of the dauer molt, washed with chase media for 60 min, and radioactivity in cuticle and noncuticle macromolecules determined for different portions of the population at subsequent times during development. The results of this experiment are presented in Fig. 4. At the completion of the 60 min wash with chase media, label is present in cuticle material but has only obtained about 50% of the maximum value reached after a further l-2 hr of chase. No comparable delay is observed for the maximum labeling of noncuticle macromolecules; therefore, the l-2 hr delay observed for the maximum labeling of cuticle material cannot be attributed to an ineffective chase. Time lags of this sort are characteristic of secretion processes (Kafatos and Kiortis, 1971) and thus would be expected for cuticle radioactivity since our isolation procedures measure only secreted and cross-linked cuticle material. During the L4 stage and early part of the L4 lethargus the amount of radioactivity in cuticle material remains fairly constant. As the nematode population proceeds through the L4 molt, however, cuticle radioactivity declines rapidly, coinciding with the resumption of pharyngeal pumping and shedding of the L4 cuticle at this molt. By the time that 90% of the population has completed the L4 molt (resumed pumping), 88% of the radioactivity maximally present in cuticle material has disappeared. The amount of radioactivity in noncuticle macromolecules, in contrast, shows little change over these same time periods. DISCUSSION

The usual methods for introducing radioactive tracers into nematode proteins have been either to incubate them in liquid solutions containing radioactive amino acids (Leushner and Pasternak, 1973) or to feed them bacteria prelabeled with 35S (Garcea et aZ., 1978). Both methods require that the nematodes be feeding for free amino acids are not readily absorbed by the worm through the cuticle or hypodermis (Marks et aZ., 1968). Since previous studies suggested that the nonfeeding lethargus periods were times of rapid cuticle synthesis we sought other means for labeling nematode proteins that did not require the label to be orally ingested. As was demonstrated, NaH14C03 proved useful for this purpose. This label has previously been used to study various aspects of protein and amino acid metabolism in both vertebrate and invertebrate systems (Awapara and Campbell, 1964; Swick and Ip, 1974). In C. elegans, NaH14C0, appears to be a relatively specific macromolecular label for amino acids in proteins. At least 75% of the radioactivity incorporated into cuticle material and at least 65% of the radioactivity incorporated into noncuticle macromolecules could be ac-

COX ET AL.

C. elegans

counted for as labeled amino acids. The identity of the remaining macromolecular radioactivity is not known; however, it does not appear to be significantly present in RNA, DNA, or as amino sugars attached to proteins. While we are somewhat uneasy with the fact that we cannot account for all of the radioactivity incorporated into cuticle and noncuticle macromolecules, we do not feel that this uncertainty vitiates the conclusions to be drawn from this work. Consistent with the known pathways of CO, fixation the major amino acids found to be labeled after exposure of C. elegans to NaH14C0, were those most closely related to the tricarboxylic acid cycle and presumably arose by transamination and equilibration with other pathways. Rothstein (1965) has demonstrated that Caenorhabditis possesses a complete tricarboxylic acid cycle and all the common transaminases necessary for these inter-conversions. The fact that similar amino acids were found to be labeled in both cuticle and noncuticle proteins suggests that these enzymes are active in several tissues of C. elegans. Because of its ability to enter the worm rapidly in the absence of oral digestion and to be effectively chased from the animal following a short pulse, NaH14C0, should prove useful for future studies of protein synthesis during molting and for studies of protein synthesis in other systems where electrolyte permeability barriers exist, as is the case for nematode eggs (Bird, 1971). The present results show that NaH14C0, is incorporated into cuticle material in a discontinuous manner during the development of C. elegans from a dauer larva to an adult. Using pulse-labeling regimens, two periods of elevated incorporation were observed and these coincided with the lethargus periods of the dauer and L4 molts. No such discontinuous pattern was found for the incorporation of NaH14C0, into total noncuticle macromolecules during development. Pulse -chase experiments confirmed that the increased incorporation of NaH14C0, into cuticle material at each molt was due to actual increases in the amount of cuticle material synthesized at these times rather than to variations in the rates of secretion or cross-linking of cuticle material during development. Since the molts were not periods of general metabolic increases, we believe these fluctuations to represent true variations in the relative rate of cuticle protein synthesis during development. Our findings are consistent with two other lines of experimental evidence. First, at each molt there is a marked accumulation of Golgi bodies and ribosomes within the hypodermal cells, suggestive of increased synthetic activity at these times (Singh and Sulston, 1978). Second, Leushner and Pasternak (1975) found that a burst of collagen synthesis precedes each ecdysis during development of P. silusiae. Our results lend strong support to their proposal that these increases were in fact

Cuticle

Synthesis

283

due to increased cuticle collagen synthesis at these times. We were able to study separately the synthesis of the protein components of the inner and outer layers of the cuticle since they could be distinguished on the basis of their solubility in the presence of a sulfhydryl reducing agent (Cox, Kusch, and Edgar, manuscript submitted). The inner layers of the cuticle are composed primarily of collagen-like proteins while the outer cuticle layers appear to be composed principally of noncollagen proteins. Both classes of cuticle proteins displayed similar temporal patterns of synthesis during the two molts examined. No difference could be detected in the periods of peak synthesis of these two groups of proteins with the 2 hr labeling intervals used in these experiments. A ratio of about 1.5 : 1 was found for the synthesis of soluble to insoluble cuticle components during the dauer molt while a slightly higher ratio of 2: 1 was found for the synthesis of soluble to insoluble cuticle components during the L4 molt. Similar ratios of these two groups of cuticle proteins have been determined for L4’s and adults continuously labeled with 35S E. coli (our unpublished results) and by weighing adult cuticle fractions directly (Cox, Kusch, and Edgar, manuscript submitted). Therefore, the synthetic ratios observed for these two groups of cuticle proteins in these experiments apparently reflect their true proportions within the L4 and adult cuticles. It is possible that a finer temporal pattern of synthesis and/or secretion of specific cuticle proteins might be revealed through a combination of radiolabeling and electrophoretic methods. Since the cuticle appears to be secreted in layers (Davey, 1965; our unpublished results), studies of these sorts might aid in the localization of specific proteins to particular zones or structures within the cuticle. The molecular mechanisms responsible for the oscillating pattern of cuticle synthesis during development remain unknown; however, the observation of Kan and Davey (1968) that the RNA content of the hypodermis undergoes cyclic increases during development in parallel with the molting cycles and the finding of Pasternak and Leushner (1975) that the increases in collagen synthesis normally preceding each ecdysis during development of P. silusiae are inhibited in the presence of agents that block messenger RNA transcription suggest that cuticle protein synthesis is modulated during development in response to changes in the rates of synthesis and accumulation of cuticle protein messenger RNAs. A definitive answer to this question must await the development of probes for the assay of cuticle protein messenger RNAs during development. The fmding that the structural components of the cuticle account for approximately 10% of the macromolecules labeled by NaHi4C03 at the lethargus peaks of the two

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VOLUME 84, 1981

molts examined suggests that cuticle synthesis occupies many of the biochemical events leading to the formation a large proportion of the worm’s metabolism at these of the L4 and adult cuticles (and presumably the cuticles times. This conclusion should be considered only tentaof the other developmental stages) of C. elegans are tive, however, for a number of technical reasons. First, quite similar. Each cuticle is apparently assembled from a set of precursors whose synthesis is largely restricted cuticle proteins are relatively enriched for the amino to the lethargus periods. At both the dauer and L4 molts acids labeled by NaH14C0, (Cox, Kusch, and Edgar, manuscript submitted) and thus their synthesis may tend a similar proportion of the worm’s metabolism appears to to be overestimated by this label. Second, it is not known be devoted to cuticle synthesis and similar ratios of soluif the activities of the CO, fixing enzymes and major ble and insoluble cuticle components are produced. Howtransaminases are the same in the various tissues of C. ever, the cuticles of the L4 and the adult (and most of the elegans. Tissue-specific difference in the activities of the other stages of C. elegans) are architecturally quite difmajor transaminases have been reported for Ascaris ferent from one another and each contains some unique (Pollak and Fairbairn, 1955). On the other hand, 10% proteins (our unpublished results). Therefore, the gemay actually be an underestimate of the true proportion netic program that controls molting cannot be a simple of cuticle-related synthesis occurring at each molt, since reiterative one but rather must be one that is capable of this value does not include the synthesis of any nonstrucdirecting and integrating a series of related processes tural proteins associated with the synthesis, processing, that contain both unique and shared events. Determinaor secretion of cuticle proteins. Further studies of the tion of how this genetic program operates should aid in molting process using other types of labeling methods the understanding of many complex developmental phemay prove helpful in resolving this question. nomena. In certain nematode species the old cuticle is not shed We would like to thank Dr. Paulo Dice for originally suggesting the intact at each molt but rather is partially resorbed and may possibly be reutilized in the formation of the new cu- use of NaH1%03 for these experiments and for providing facilities during the stay of one of us (K.D.) in his laboratory. This work was sup ticle (Bird and Rogers, 1965; Johnson et al., 1970). This ported by grants PCM%-11481 and PCM78-09439 from the National does not appear to be the case for C. elegans. From elec- Science Foundation and grant 5-S0’7RR07135 from the National Institron microscopy studies (Singh and Sulston, 1978; our tutes of Health. unpublished results), it appears that C. elegans sheds its old cuticle intact at each molt. The pulse-chase experiREFERENCES ments reported here support this conclusion. We failed AWAPARA, J., and CAMPBELL, J. W. (1964). Utilization of %Oa for the to detect any significant incorporation of NaH14C0, into formation of some amino acids in three invertebrates. Comp. adult cuticle components during periods of maximum L4 B&hem. Physiol. 11, 231-235. cuticle synthesis. In addition, we showed that at least BIRD, A. F. (1971). “The Structure of Nematodes.” Academic Press, 88% of the radioactivity incorporated into L4 cuticle maNew York. terial during the dauer molt was subsequently lost at the BIRD, A. F. and ROGERS, G. E. (1965). Ultrastructure of the cuticle and its formation in Meloidogyne javanica. Nematologica 11, 224next ecdysis. 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