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Dauer larva recovery in the nematode Caenorhabditis elegans—I. The effect of mRNA synthesis inhibitors on recovery, growth and pharyngeal pumping
Comp. Biochem. Physiol. Vol. 98B, No. 2/3, pp. 239-243, 1991 Printed in Great Britain
0305-0491/91 $3.00+ 0.00 © 1991 PergamonPressplc
DAUER LARVA RECOVERY IN THE NEMATODE CAENORHABDITIS E L E G A N S - - I. THE EFFECT OF mRNA SYNTHESIS INHIBITORS ON RECOVERY, GROWTH A N D PHARYNGEAL PUMPING THERESA J. REAPE and ANN M. BURNELL* Department of Biology, St Patrick's College, Maynooth, Co. Kildare, Ireland (Tel: 353-1-6285222)
(Received 11 July 1990) Abstract--l. There are several well-characterized stages in the recovery of dauer larvae, beginning with resumption of pharyngeal pumping and followed by growth of the dauer larvae to normal juvenile diameter. 2. The mRNA synthesis inhibitors actinomycin D and ~-amanitin failed to affect these critical components of the recovery process. 3. Polyacrylamide gel electrophoresis revealed the loss of dauer specific bands in the presence of high actinomycin D concentrations, an indication of normal exit from the dauer state. 4. These results indicate that mRNA synthesis is not required for the initial pre-molt period of dauer larva recovery, but failure of the worms to molt in the presence of actinomycin D or ~-amanitin suggests that mRNA synthesized during the recovery period is required for the first post-dauer molt.
INTRODUCTION Under favourable growth conditions of low population density and abundant food supply, newly hatched larvae of the free living nematode Caenorhabditis elegans undergo four larval stages (L1 through L4) in approximately 2.5 days at 20°C before becoming adults (Byerly et al., 1976). When adverse environmental conditions interfere with normal development, juveniles of C. elegans can enter a non-feeding form specialized for survival and dispersal, the dauer larva. Dauer larvae have several distinctive properties which have been described by Cassada and Russell (1975). They are thin, possess an altered cuticle structure and pharyngeal pumping is completely suppressed making them more resistant to harsh conditions and various detergents such as sodium dodecyl sulphate (SDS). Functional glycolytic, gluconeogenetic, tricarboxylic acid cycle and oxidative phosphorylation pathways are present in the dauer larva of C. elegans but with reduced activity relative to adults (O'Riordan and Bumell, 1989). This reduced metabolic rate would appear to form part of the adaptive response which enables dauer larvae to survive for several months without feeding. When dauer larvae are transferred to fresh food medium a sequence of events occurs, termed recovery, whereby they resume feeding and molt to become 1..4 larvae and apparently fully rejoin the normal developmental pathway. Once pharyngeal pumping is resumed, the worms lose their resistance to SDS. The dauer specific cuticle is lost during the first postrecovery molt which occurs approximately 13 hr after *Author to whom correspondence should be addressed.
introduction to food. During the initial stages of recovery, worms swell to normal juvenile diameter (Cassada and Russell, 1975) and limited longitudinal growth can be observed after 4 hr (see Fig. 1). However, rapid longitudinal growth does not occur until after the first post-recovery molt. A fatty acid-like pheromone, produced by densely populated cultures of worms, appears to serve as a measure of population density and high pheromone concentrations induce dauer larva formation and prevent recovery (Golden and Riddle, 1982; Ohba and Ishibashi, 1982). F o o d acts in competition with the pheromone to enhance recovery. Since dauer larvae exists in a repressed metabolic state, the transition from the quiescent dauer stage to an active feeding stage must require a switch in the developmental programme. Wadsworth and Riddle (1988) have reported an abundance of ATP and other high energy metabolites within 6 hr of introduction of dauer larvae to a food source, an indication of entry into a more active metabolic state. In the investigations reported here, antibiotics with known sites of action at the level of transcription and translation were employed in order to ascertain if these processes are important for dauer larva recovery. This type of approach has been used on a wide range of organisms to determine whether protein or m R N A synthesis are required for a certain developmental process (e.g. Widelitz et al., 1986; Scadding, 1988; Maharajan el al., 1989; Otani et al., 1989). This paper describes the results obtained using m R N A synthesis inhibitors and an accompanying paper (Reape and Burnell, 1991) documents the effect of protein synthesis inhibitors on dauer recovery and on growth and pharyngeal pumping in recovering C. elegans dauer larvae.
239
THERESA J. REAPE and ANN M. BURN'ELL
240
MATERIALS AND METHODS
Maintenance and growth of the organism The C. elegans stock used in this work was the N2 Bristol strain. Nematodes were grown monoxenically in mass culture using E. cod strain OP50 as a food source as described by Sulston and Brenner (1974). E. coli was resuspended in S medium at a concentration of 1% w/v and 75 ml of this medium was added to 250 ml flasks and innoculated with approximately 1 ml of a 3-week-old culture containing mostly dauer larvae. The cultures were incubated at 20°C on a rotary shaker at 150 rev/min for 3 weeks.
Isolation of dauer larvae from mixed populations Three-week-old liquid cultures of worms were centrifuged at 1400g for 15min on a Sorval RC-5B centrifuge. The pelleted nematodes were washed several times using distilled water and resuspended in cold 35% w/v sucrose and immediately centrifuged in a swinging bucket at 1549 g for 15 rain on a Heraeus refrigerated bench centrifuge in order to separate worms from remaining bacteria. Nematodes were washed again with distilled water and treated with 1% SDS for 1 hr (Cassada and Russell, 1975). The surviving dauer larvae and carcasses were then washed thoroughly and centrifuged through a cushion of cold 15% Ficoll at 300g for 10 min (Cox et al., 1981). The pelleted dauer larvae were washed several times and used for experimental work immediately after isolation.
Preparation of worms for recovery assays A series of 100 ml Erlenmeyer flasks containing 20 ml of S medium and E. eoli plus the appropriate concentration of antibiotic were innoculated with an equal volume of dauer larvae, in M9 buffer (Brenner, 1974). Control flasks were also set up containing the same recovery medium minus the antibiotic. Flasks were aerated on a rotary shaker at 20°C.
Influence of actinomycin D and o~-amanitin on growth of recovering dauer larvae A range of actinomycin D (2.5-20 pM) and ~,-amanitin (5-30 #M) concentrations were assayed for their effect on recovering dauer larvae. At various time intervals aliquots of worms were placed on a microscope slide and immobilized and straightened by heat killing. This procedure involved passing the end of the slide, containing the worms, through the flame of a bunsen for a few seconds. The lengths of the worms were then recorded in mm using an Olympus microscope fitted with a micrometer.
were also introduced into recovery medium without actinomycin D and this represented the control. After 12hr, worms were washed with distilled water and floated in 35% sucrose. Worms were then washed several times in distilled water and left for 30 rain in order to digest any bacteria remaining in the gut. A final wash was carried out in 0.0625 M Tris-HC1 buffer. Worms were then sonicated in 0.2 ml buffer for 30 sec.
Gel electrophoresis PAGE was performed by the method of Laemmli (1970) with 10% acrylamide gels. Protein (20#g) was added to each lane. After electrophoresis, the gels were stained with 0.25% Coomassie blue R-250 in 5% methanol-10% TCA-7.5% acetic acid and destained in 10% methanol-15% acetic acid. Protein concentrations were estimated using the Bio-Rad Protein Assay Kit (first demonstrated by Bradford, 1976) using bovine serum albumin as the standard. RESULTS
1. The effects o f cycloheximide and aetinomycin D on dauer larva recovery Pharyngeal p u m p i n g a n d grazing m o t i o n s of the h e a d are suppressed in d a u e r larvae. Once p h a r y n g e a l p u m p i n g has resumed in recovering animals, they become sensitive to SDS. SDS sensitivity a n d p h a r y n geal p u m p i n g assays were carried o u t in order to d e m o n s t r a t e the effects o f increasing c o n c e n t r a t i o n s o f actinomycin D o n these central c o m p o n e n t s o f the recovery process. The effect o f this drug o n longitudinal growth was also monitored. Actinomycin D is a n antibiotic t h a t acts by binding to D N A , so as to selectively inhibit the capacity o f D N A to serve as a template for R N A synthesis (Ernst a n d Oleinick, 1977). 1.1. Growth. A c t i n o m y c i n D was f o u n d to retard the g r o w t h o f recovering d a u e r larvae in a dosed e p e n d e n t m a n n e r (Fig. 1). T h e lower c o n c e n t r a t i o n s tested slowed d o w n g r o w t h b u t the w o r m s ultimately rejoined the n o r m a l developmental pathway. A 1.2 - -
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SDS sensitivity assays In this assay the sensitivity of recovering dauer larvae to a 1% SDS solution was monitored. Aliquots of worms were pipetted into a 24-well plate at hourly intervals after introduction to food and treated with 1% SDS for 30 min. The worms were then washed free of SDS and transferred to fresh NG agar plates seeded with E. coli (Cassada and Russell, 1975). Survivors were scored 24 hr later.
Pumping assays At hourly intervals, an aliquot of the culture to be examined (i.e. containing both nematodes and food source) was pipetted onto a microscope slide and covered with a clean coverslip, in order to slow down worm movement. The worms were then monitored in turn for pharyngeal pumping for 5 sec using an Olympus monocular microscope at × 100 magnification.
Preparation of samples for SDS-PAGE (polyacrylamide gel electrophoresis) Dauer larvae were added to S medium containing E. coli and incubated at 20°C for 12 hr in the presence of 10 and 20 # M actinomycin D, i.e. concentrations of the drug which retarded or prevented growth, respectively. Dauer larvae
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Fig. l. The effect of actinomycin D on longitudinal growth of recovering dauer larva: 2.5 (ll); 5 (O); 7.5 ([-]); 10 (A) and 2 0 # M (V), as compared with growth of normal recovering dauers (Q).
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Fig. 2. The effect of increasing concentrations of actinomycin D on resumption of pharyngeal pumping in recovering dauer larvae: control (e); 2.5 (ll); 5 (©); 7.5 ([2); 10 (A) and 20 #M (A). concentration 20 # M actinomycin D prevented the animals from molting and thus no further longitudinal growth occurred. The drug did not affect worm motility. 1.2. Pharyngeal pumping. Pumping was monitored in recovering dauer larvae in a range of actinomycin D concentrations. High concentrations of actinomycin D had little effect on resumption of pumping as can be seen from Fig. 2. Six hours after introduction to food 92% of the dauer larvae, treated with 20 # M actinomycin D, were pumping compared with 97% of the control worms. 1.3. SDS resistance. The SDS resistance of dauer larvae depends both on their tightly closed mouth, their lack of pumping and on their special cuticle. However, once a pumping sequence is initiated, recovering dauers are rapidly killed by the internal detergent action of SDS (Cassada and Russell, 1975). The results obtained for the SDS sensitivity assays are presented in Fig. 3, from which it can be seen that daucr larvae become sensitive to SDS when actinomycin D was present in the recovery medium. This finding implies that m R N A synthesis is unlikely to be an essential component of the recovery process.
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Fig. 3. The effect of 15 (A) and 20 #M (V) actinomycin D on SDS sensitivity in recovering dauer larvae as compared with control larvae (O). Fifteen/~M actinomycin D was tested for its effect on both growth and resumption of pharyngeal pumping in recovering dauer larvae, but as results were very similar to those obtained for 20/zM the data are not presented in Figs 1 and 2. However, these authors found that a concentration of 20/~g/ml (22.1/tM) was required to prevent longitudinal growth of L1 larvae. Nematode growth in recovery medium in the presence of =-amanitin was inhibited in a dose-dependent manner (Fig. 4). However, even at 30/zM, a concentration which completely prevented longitudinal growth in recovering dauer larvae, 93% of the worms monitored were pumping after 4 h r in recovery medium as compared with 90% of the controls 1.2
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The results obtained using actinomycin D implied that m R N A synthesis is not required for entry into the recovery phase. This was tested further using ~,amanitin, an inhibitor of R N A polymerase II (Lindell et al., 1970). Sanford et al. (1983) have demonstrated that =-amanitin at a concentration of 6.7 ng/ml (7.47 nM) resulted in 50% inhibition of R N A polymerase II activity in a mixed population of C. elegans.
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242
Tr~.RmAJ. REAPEand ANN M. BURN'ELL
Table 1. Effectof ~t-amanitinon resumptionof pharyngealpumpihg in recoveringdauer larvae* Time (hr) Control 10;tM 20/zM 30#M 0 0 0 0 0 1 43 12 34 27 2 36 30 35 14 3 28 25 74 74 4 90 77 81 93 5 94 93 84 93 6 90 92 97 93 7 90 92 92 97 *Each valuerepresentsthe percentageof wormspumpingout of a total of 50 observations. Each valuehas beenroundedoff to the nearest wholenumber. (Table 1). This result, taken in combination with that obtained for actinomycin D, provides strong evidence that neither mRNA synthesis nor new gene expression is required for exit from the dauer stage. 3. P A G E analysis SDS-PAGE was carried out and the gels were stained with Coommassie blue in order to show quantitative changes occurring in protein patterns in recovering dauer larvae. Lane 5 (Fig. 5) shows the protein pattern obtained for the E. coli food stock used in the recovery medium. Comparison of lane 5 with lanes 2-4 shows that dauers were successfully washed free of E. coli since none of the major bacterial protein bands are evident in the lanes containing the nematode extracts. Four main bands A - D of molecular weights 73,000, 64,000, 47,000 and 41,000, respectively, can be seen in the dauer extracts (lane 1). After 12 hr in recovery medium, the most noticeable feature in the control extract is the loss of band A and the reduction in intensity of bands B and D. The loss of band A and the reduction in intensity of bands B and D also occurs in the actinomycin D treated recovering dauer larvae. This result also indicates that the recovery process is not affected by the presence of actinomycin D in the recovery medium. Although the number of proteins detected on these Coomassie blue-stained gels is low, the results suggest that dauer recovery is associated with the loss of major dauer proteins, in particular bands A and D. Since the extracts used in the gel were isolated before the first post-dauer moult had occurred, bands A and D are unlikely to be dauer specific cuticular proteins. Further, the loss of band A and the reduction of band D also occurs in actinomycin D-treated recovering dauers where the post dauer moult is suppressed.
expression is necessary for this stage of the recovery process, ct-Amanitin, which affects RNA synthesis through a different mechanism to actinomycin D was found to have similar affects on growth and pharyngeal pumping (Fig. 4 and Table 1). PAGE performed after 12 hr of recovery and stained with Coomassie blue to show quantitative changes, showed several dauer bands, i.e. 73,000, 64,000 and 41,000 mol. wts, diminishing in intensity, in both actinomycin D treated and control larvae (Fig. 5), an indication that exit from the dauer stage can occur in the presence of actinomycin D. Riddle et al. (1981) have observed that there are about 100 genes affecting dauer formation but that few genes, if any, are involved specifically in the exit from the dauer stage and they hypothesized that the genes required for dauer recovery may be a subset of the genes which are required for entry into the dauer stage. It is perhaps not suprising then that mRNA synthesis is not required for exit from the dauer stage to occur. Rather, it seems more likely that prior to or during the dauer larval stage, the worms synthesize and store a sufficient amount of mRNA which they will require if they perceive the signal to initiate a recovery sequence and that the proteins necessary for recovery are in turn synthesized from this existing mRNA. In contrast to prokaryotes where mRNA molecules exist for only a few minutes, the mRNA in eukaryotes is relatively stable. In mammalian cells, estimates of half-lifes of long-lived mRNAs vary from 24 to 100 hr, depending on the system (Singer and Penman, 1973). The rates of mRNA degradation can change dramatically in response to some biological or pharmacological stimuli, e.g. vitellogenin mRNA was
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DISCUSSION Actinomycin D did not interfere with resumption of pharyngeal pumping and worms became sensitive to SDS (Figs 2 and 3) during the early stages of recovery. Very high concentrations of the drug which completely prevented growth had little effect on pumping and although worms did swell slightly in diameter from intake of food, no longitudinal growth was observed. The failure of actinomycin D to affect dauer larva recovery indicates the mRNA synthesis is not required for exit from the dauer stage. However, failure of the worms to molt in the presence of high concentrations of the drug indicate that new gene
Fig. 5. Stained patterns of proteins from dauer larvae and dauers which had been in recovery medium for 12 hr in the presence and absence of actinomycin D. Proteins were separated on a 10% polyacrylamide gel and stained with Coomassie blue. 20/~g of protein extract was applied to each lane. Lane 1 (dauer), 2 (control), 3 (10#M act D), 4 (20 #M act D) and 5 (E. coli OP50, the food source used in the recovery medium).
Caenorhabditis dauer larva recovery--I
shown to be degraded with a half-life of 480 hr in oestrogen-treated cells compared to a half life of 16 hr in untreated cells (Brock and Shapiro, 1983). Similarly, histone m R N A is stable for months in Xenopus oocyctes but is degraded within hours upon fertilization (Ruderman et al., 1979; W o o d l a n d et al., 1979). Thus, our finding that m R N A synthesis seems not to be obligatory for dauer recovery suggests that the dauer larva may represent a developmental state analogous to unfertilized oocytes which store maternal m R N A s for long periods (reviewed by Davidson, 1986) or to diapausing insect embryos which contain a store of inactive m R N A s which are activated during post diapause development (see Saito et al., 1985, for example). Translation of stored m R N A may perhaps be triggered when dauer larvae perceive a food signal. If this is the case then protein synthesis inhibitors might be effective in preventing dauer recovery. In all accompanying paper (Reape and Burnell, 1991) we present the results obtained in an investigation of the effect of inhibitors of protein synthesis on dauer larva recovery. REFERENCES
Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Brenner S. (1974) The genetics of Caenorhabditis elegans. Genetics 77, 71-94, Brock M. L. and Shapiro D. J. (1983) Estrogen stabilizes vitellogenin mRNA against cytoplasmic degradation. Cell 34, 207-214. Byerly L., Cassada R. C. and Russell R. L. (1976) The life-cycle of the nematode Caenorhabditis elegans. I. Wild type growth and reproduction. Dev. Biol. 51, 24-33. Cassada R. C. and Russell R. L. (1975) The dauer larva, a post-embryonic developmental variant of the nematode Caenorhabditis elegans. Dev. Biol. 46, 326-342. Cox G. N., Staprans S. and Edgar R. S. (1981) The cuticle of Caenorhabditis elegans II. Stage-specificchanges in ultrastructure and protein composition during postembryonic development. Dev. Biol. 86, 456-470. Davidson E. H. (1986) In Gene Activity in Early Development, 3rd edn, pp. 69-106 and pp. 173-187. Academic Press, Orlando, FL. Ernst S. G. and Oleinick N. L. (1977) Actinomycln D in Tetrahymena. Non-specific inhibition of RNA synthesis and primary and secondary effects on protein synthesis. Exp. Cell. Res. 110, 363-373. Golden J. W. and Riddle D. L. (1982) A pheromone influences larval development in the nematode Caenorhabditis elegans. Science 218, 578-580. Golden J. W. and Riddle D. L. (1984) The Caenorhabditis elegans dauer larva: developmental effects of pheromone food and temperature. Dev. Biol. 102, 368-378. Laemmli M. K. (1970) Cleavage of structural proteins during the assembly of the head of bacterophage T4. Nature 227, 680-685.
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Lindell T. J., Weinberg F., Morris P. W., Roeder R. G. and Rutter W. J. 0970) Specific inhibition of nuclear RNA polymerase II by a-amanitin. Science 170, 447-449. Maharajan P., Gargiulo C. and Maharajan V. (1989) Stage specific inhibition of sea urchin development by ct-amanitin. Invert. Reprod. Dev. 15, 13-18. Ohba K. and Ishibashi N. (1982) A factor inducing dauer juvenile formation in Caenorhabditis elegans. Nematologica 28, 318-325. O'Riordon V. B. and Burnell A. M. (1989) Intermediary metabolism in the dauer larva of the nematode Caenorhabditis elegans--I. Glycolysis, gluconeogenesis, oxidative phosphorylation and the tricarboxylic acid cycle. Comp. Biochem. Physiol. 92B, 233-238. Otani S., Marshall W. P., Tate W. P., Goddard G. V. and Abraham W. C. (1989) Maintenance of long-term potentiation in rat dentate gyrus requires protein synthesis but not messenger RNA synthesis immediately post-tetanization. Neuroscience 28, 519-526. Reape T. J. and Burnell A. M. (1991) Dauer larva recovery in the nematode Caenorhabditis elegans--II. The effect of inhibitors of protein synthesis on recovery, growth and pharyngeal pumping. Comp. Biochem. Physiol. 98B, 245-252. Riddle D. L., Swanson M. M. and Albert P. S. (1981) Interacting genes in nematode dauer larva formation. Nature 290, 668-671. Ruderman J. V., Woodland H. R. and Sturgess E. A. (1979) Modulations of histone messenger RNA during the early development of Xenopus laevis. Dev. Biol. 71, 71-82. Saito A., Sugimoto Y., Koga K. and Sakaguchi B. (1985) Translation of RNA from eggs during post-diapause development of Bombyx mori. Develop. Growth. Differ. 27, 13-20. Sanford T., Golomb M. and Riddle D. L. (1983) RNA polymerase II from wild type and ~-amanitin-resistant strains of Caenorhabditis elegans. J. biol. Chem. 258, 12804-12809. Scadding R. S. (1988) Actinomycln D, cycloheximide and tunicamycln inhibit vitamin A induced proximodistal duplication during limb regeneration in the axolotl Ambystoma mexicanum. Can. J. Zool. 66, 879-884. Singer R. H. and Penman S. (1973) Messenger RNA in HeLa cells: kinetics of formation and decay. J. molec. Biol. 78, 321-334. Sulston J. E. and Brenner S. (1974) The DNA of Caenorhabditis elegans. Genetics 77, 95-104. Wadsworth W. G. and Riddle D. L. (1988) Acidic intracellular pH shift during Caenorhabditis elegans larval development. Proc. natn. Acad. Sci. U.S.A. 85, 8435-8438. Widelitz R. B., Magun B. E. and Gerner E. W. (1986) Effects of cycloheximide on thermotolerance expression heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts. Mol. Cell. Biol. 6, 1088-1094. Woodland H. R., Flynn J. M. and Wyllie A. J. (1979) Utilization of stored mRNA in Xenopus embryos and its replacement by newly synthesised transcripts: histone H1 synthesis using interspecles hybrids. Cell 18, 165-171.
0305-0491/91 $3.00+ 0.00 © 1991 PergamonPressplc
DAUER LARVA RECOVERY IN THE NEMATODE CAENORHABDITIS E L E G A N S - - I. THE EFFECT OF mRNA SYNTHESIS INHIBITORS ON RECOVERY, GROWTH A N D PHARYNGEAL PUMPING THERESA J. REAPE and ANN M. BURNELL* Department of Biology, St Patrick's College, Maynooth, Co. Kildare, Ireland (Tel: 353-1-6285222)
(Received 11 July 1990) Abstract--l. There are several well-characterized stages in the recovery of dauer larvae, beginning with resumption of pharyngeal pumping and followed by growth of the dauer larvae to normal juvenile diameter. 2. The mRNA synthesis inhibitors actinomycin D and ~-amanitin failed to affect these critical components of the recovery process. 3. Polyacrylamide gel electrophoresis revealed the loss of dauer specific bands in the presence of high actinomycin D concentrations, an indication of normal exit from the dauer state. 4. These results indicate that mRNA synthesis is not required for the initial pre-molt period of dauer larva recovery, but failure of the worms to molt in the presence of actinomycin D or ~-amanitin suggests that mRNA synthesized during the recovery period is required for the first post-dauer molt.
INTRODUCTION Under favourable growth conditions of low population density and abundant food supply, newly hatched larvae of the free living nematode Caenorhabditis elegans undergo four larval stages (L1 through L4) in approximately 2.5 days at 20°C before becoming adults (Byerly et al., 1976). When adverse environmental conditions interfere with normal development, juveniles of C. elegans can enter a non-feeding form specialized for survival and dispersal, the dauer larva. Dauer larvae have several distinctive properties which have been described by Cassada and Russell (1975). They are thin, possess an altered cuticle structure and pharyngeal pumping is completely suppressed making them more resistant to harsh conditions and various detergents such as sodium dodecyl sulphate (SDS). Functional glycolytic, gluconeogenetic, tricarboxylic acid cycle and oxidative phosphorylation pathways are present in the dauer larva of C. elegans but with reduced activity relative to adults (O'Riordan and Bumell, 1989). This reduced metabolic rate would appear to form part of the adaptive response which enables dauer larvae to survive for several months without feeding. When dauer larvae are transferred to fresh food medium a sequence of events occurs, termed recovery, whereby they resume feeding and molt to become 1..4 larvae and apparently fully rejoin the normal developmental pathway. Once pharyngeal pumping is resumed, the worms lose their resistance to SDS. The dauer specific cuticle is lost during the first postrecovery molt which occurs approximately 13 hr after *Author to whom correspondence should be addressed.
introduction to food. During the initial stages of recovery, worms swell to normal juvenile diameter (Cassada and Russell, 1975) and limited longitudinal growth can be observed after 4 hr (see Fig. 1). However, rapid longitudinal growth does not occur until after the first post-recovery molt. A fatty acid-like pheromone, produced by densely populated cultures of worms, appears to serve as a measure of population density and high pheromone concentrations induce dauer larva formation and prevent recovery (Golden and Riddle, 1982; Ohba and Ishibashi, 1982). F o o d acts in competition with the pheromone to enhance recovery. Since dauer larvae exists in a repressed metabolic state, the transition from the quiescent dauer stage to an active feeding stage must require a switch in the developmental programme. Wadsworth and Riddle (1988) have reported an abundance of ATP and other high energy metabolites within 6 hr of introduction of dauer larvae to a food source, an indication of entry into a more active metabolic state. In the investigations reported here, antibiotics with known sites of action at the level of transcription and translation were employed in order to ascertain if these processes are important for dauer larva recovery. This type of approach has been used on a wide range of organisms to determine whether protein or m R N A synthesis are required for a certain developmental process (e.g. Widelitz et al., 1986; Scadding, 1988; Maharajan el al., 1989; Otani et al., 1989). This paper describes the results obtained using m R N A synthesis inhibitors and an accompanying paper (Reape and Burnell, 1991) documents the effect of protein synthesis inhibitors on dauer recovery and on growth and pharyngeal pumping in recovering C. elegans dauer larvae.
239
THERESA J. REAPE and ANN M. BURN'ELL
240
MATERIALS AND METHODS
Maintenance and growth of the organism The C. elegans stock used in this work was the N2 Bristol strain. Nematodes were grown monoxenically in mass culture using E. cod strain OP50 as a food source as described by Sulston and Brenner (1974). E. coli was resuspended in S medium at a concentration of 1% w/v and 75 ml of this medium was added to 250 ml flasks and innoculated with approximately 1 ml of a 3-week-old culture containing mostly dauer larvae. The cultures were incubated at 20°C on a rotary shaker at 150 rev/min for 3 weeks.
Isolation of dauer larvae from mixed populations Three-week-old liquid cultures of worms were centrifuged at 1400g for 15min on a Sorval RC-5B centrifuge. The pelleted nematodes were washed several times using distilled water and resuspended in cold 35% w/v sucrose and immediately centrifuged in a swinging bucket at 1549 g for 15 rain on a Heraeus refrigerated bench centrifuge in order to separate worms from remaining bacteria. Nematodes were washed again with distilled water and treated with 1% SDS for 1 hr (Cassada and Russell, 1975). The surviving dauer larvae and carcasses were then washed thoroughly and centrifuged through a cushion of cold 15% Ficoll at 300g for 10 min (Cox et al., 1981). The pelleted dauer larvae were washed several times and used for experimental work immediately after isolation.
Preparation of worms for recovery assays A series of 100 ml Erlenmeyer flasks containing 20 ml of S medium and E. eoli plus the appropriate concentration of antibiotic were innoculated with an equal volume of dauer larvae, in M9 buffer (Brenner, 1974). Control flasks were also set up containing the same recovery medium minus the antibiotic. Flasks were aerated on a rotary shaker at 20°C.
Influence of actinomycin D and o~-amanitin on growth of recovering dauer larvae A range of actinomycin D (2.5-20 pM) and ~,-amanitin (5-30 #M) concentrations were assayed for their effect on recovering dauer larvae. At various time intervals aliquots of worms were placed on a microscope slide and immobilized and straightened by heat killing. This procedure involved passing the end of the slide, containing the worms, through the flame of a bunsen for a few seconds. The lengths of the worms were then recorded in mm using an Olympus microscope fitted with a micrometer.
were also introduced into recovery medium without actinomycin D and this represented the control. After 12hr, worms were washed with distilled water and floated in 35% sucrose. Worms were then washed several times in distilled water and left for 30 rain in order to digest any bacteria remaining in the gut. A final wash was carried out in 0.0625 M Tris-HC1 buffer. Worms were then sonicated in 0.2 ml buffer for 30 sec.
Gel electrophoresis PAGE was performed by the method of Laemmli (1970) with 10% acrylamide gels. Protein (20#g) was added to each lane. After electrophoresis, the gels were stained with 0.25% Coomassie blue R-250 in 5% methanol-10% TCA-7.5% acetic acid and destained in 10% methanol-15% acetic acid. Protein concentrations were estimated using the Bio-Rad Protein Assay Kit (first demonstrated by Bradford, 1976) using bovine serum albumin as the standard. RESULTS
1. The effects o f cycloheximide and aetinomycin D on dauer larva recovery Pharyngeal p u m p i n g a n d grazing m o t i o n s of the h e a d are suppressed in d a u e r larvae. Once p h a r y n g e a l p u m p i n g has resumed in recovering animals, they become sensitive to SDS. SDS sensitivity a n d p h a r y n geal p u m p i n g assays were carried o u t in order to d e m o n s t r a t e the effects o f increasing c o n c e n t r a t i o n s o f actinomycin D o n these central c o m p o n e n t s o f the recovery process. The effect o f this drug o n longitudinal growth was also monitored. Actinomycin D is a n antibiotic t h a t acts by binding to D N A , so as to selectively inhibit the capacity o f D N A to serve as a template for R N A synthesis (Ernst a n d Oleinick, 1977). 1.1. Growth. A c t i n o m y c i n D was f o u n d to retard the g r o w t h o f recovering d a u e r larvae in a dosed e p e n d e n t m a n n e r (Fig. 1). T h e lower c o n c e n t r a t i o n s tested slowed d o w n g r o w t h b u t the w o r m s ultimately rejoined the n o r m a l developmental pathway. A 1.2 - -
1.1
SDS sensitivity assays In this assay the sensitivity of recovering dauer larvae to a 1% SDS solution was monitored. Aliquots of worms were pipetted into a 24-well plate at hourly intervals after introduction to food and treated with 1% SDS for 30 min. The worms were then washed free of SDS and transferred to fresh NG agar plates seeded with E. coli (Cassada and Russell, 1975). Survivors were scored 24 hr later.
Pumping assays At hourly intervals, an aliquot of the culture to be examined (i.e. containing both nematodes and food source) was pipetted onto a microscope slide and covered with a clean coverslip, in order to slow down worm movement. The worms were then monitored in turn for pharyngeal pumping for 5 sec using an Olympus monocular microscope at × 100 magnification.
Preparation of samples for SDS-PAGE (polyacrylamide gel electrophoresis) Dauer larvae were added to S medium containing E. coli and incubated at 20°C for 12 hr in the presence of 10 and 20 # M actinomycin D, i.e. concentrations of the drug which retarded or prevented growth, respectively. Dauer larvae
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Fig. l. The effect of actinomycin D on longitudinal growth of recovering dauer larva: 2.5 (ll); 5 (O); 7.5 ([-]); 10 (A) and 2 0 # M (V), as compared with growth of normal recovering dauers (Q).
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Fig. 2. The effect of increasing concentrations of actinomycin D on resumption of pharyngeal pumping in recovering dauer larvae: control (e); 2.5 (ll); 5 (©); 7.5 ([2); 10 (A) and 20 #M (A). concentration 20 # M actinomycin D prevented the animals from molting and thus no further longitudinal growth occurred. The drug did not affect worm motility. 1.2. Pharyngeal pumping. Pumping was monitored in recovering dauer larvae in a range of actinomycin D concentrations. High concentrations of actinomycin D had little effect on resumption of pumping as can be seen from Fig. 2. Six hours after introduction to food 92% of the dauer larvae, treated with 20 # M actinomycin D, were pumping compared with 97% of the control worms. 1.3. SDS resistance. The SDS resistance of dauer larvae depends both on their tightly closed mouth, their lack of pumping and on their special cuticle. However, once a pumping sequence is initiated, recovering dauers are rapidly killed by the internal detergent action of SDS (Cassada and Russell, 1975). The results obtained for the SDS sensitivity assays are presented in Fig. 3, from which it can be seen that daucr larvae become sensitive to SDS when actinomycin D was present in the recovery medium. This finding implies that m R N A synthesis is unlikely to be an essential component of the recovery process.
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Fig. 3. The effect of 15 (A) and 20 #M (V) actinomycin D on SDS sensitivity in recovering dauer larvae as compared with control larvae (O). Fifteen/~M actinomycin D was tested for its effect on both growth and resumption of pharyngeal pumping in recovering dauer larvae, but as results were very similar to those obtained for 20/zM the data are not presented in Figs 1 and 2. However, these authors found that a concentration of 20/~g/ml (22.1/tM) was required to prevent longitudinal growth of L1 larvae. Nematode growth in recovery medium in the presence of =-amanitin was inhibited in a dose-dependent manner (Fig. 4). However, even at 30/zM, a concentration which completely prevented longitudinal growth in recovering dauer larvae, 93% of the worms monitored were pumping after 4 h r in recovery medium as compared with 90% of the controls 1.2
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The results obtained using actinomycin D implied that m R N A synthesis is not required for entry into the recovery phase. This was tested further using ~,amanitin, an inhibitor of R N A polymerase II (Lindell et al., 1970). Sanford et al. (1983) have demonstrated that =-amanitin at a concentration of 6.7 ng/ml (7.47 nM) resulted in 50% inhibition of R N A polymerase II activity in a mixed population of C. elegans.
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Fig. 4. The effect of =-am~titin on longitudinal growth of recovering dauer larvae: control (Q); 5 (O); 10 (11); 20 if-l) and 30pM (A).
242
Tr~.RmAJ. REAPEand ANN M. BURN'ELL
Table 1. Effectof ~t-amanitinon resumptionof pharyngealpumpihg in recoveringdauer larvae* Time (hr) Control 10;tM 20/zM 30#M 0 0 0 0 0 1 43 12 34 27 2 36 30 35 14 3 28 25 74 74 4 90 77 81 93 5 94 93 84 93 6 90 92 97 93 7 90 92 92 97 *Each valuerepresentsthe percentageof wormspumpingout of a total of 50 observations. Each valuehas beenroundedoff to the nearest wholenumber. (Table 1). This result, taken in combination with that obtained for actinomycin D, provides strong evidence that neither mRNA synthesis nor new gene expression is required for exit from the dauer stage. 3. P A G E analysis SDS-PAGE was carried out and the gels were stained with Coommassie blue in order to show quantitative changes occurring in protein patterns in recovering dauer larvae. Lane 5 (Fig. 5) shows the protein pattern obtained for the E. coli food stock used in the recovery medium. Comparison of lane 5 with lanes 2-4 shows that dauers were successfully washed free of E. coli since none of the major bacterial protein bands are evident in the lanes containing the nematode extracts. Four main bands A - D of molecular weights 73,000, 64,000, 47,000 and 41,000, respectively, can be seen in the dauer extracts (lane 1). After 12 hr in recovery medium, the most noticeable feature in the control extract is the loss of band A and the reduction in intensity of bands B and D. The loss of band A and the reduction in intensity of bands B and D also occurs in the actinomycin D treated recovering dauer larvae. This result also indicates that the recovery process is not affected by the presence of actinomycin D in the recovery medium. Although the number of proteins detected on these Coomassie blue-stained gels is low, the results suggest that dauer recovery is associated with the loss of major dauer proteins, in particular bands A and D. Since the extracts used in the gel were isolated before the first post-dauer moult had occurred, bands A and D are unlikely to be dauer specific cuticular proteins. Further, the loss of band A and the reduction of band D also occurs in actinomycin D-treated recovering dauers where the post dauer moult is suppressed.
expression is necessary for this stage of the recovery process, ct-Amanitin, which affects RNA synthesis through a different mechanism to actinomycin D was found to have similar affects on growth and pharyngeal pumping (Fig. 4 and Table 1). PAGE performed after 12 hr of recovery and stained with Coomassie blue to show quantitative changes, showed several dauer bands, i.e. 73,000, 64,000 and 41,000 mol. wts, diminishing in intensity, in both actinomycin D treated and control larvae (Fig. 5), an indication that exit from the dauer stage can occur in the presence of actinomycin D. Riddle et al. (1981) have observed that there are about 100 genes affecting dauer formation but that few genes, if any, are involved specifically in the exit from the dauer stage and they hypothesized that the genes required for dauer recovery may be a subset of the genes which are required for entry into the dauer stage. It is perhaps not suprising then that mRNA synthesis is not required for exit from the dauer stage to occur. Rather, it seems more likely that prior to or during the dauer larval stage, the worms synthesize and store a sufficient amount of mRNA which they will require if they perceive the signal to initiate a recovery sequence and that the proteins necessary for recovery are in turn synthesized from this existing mRNA. In contrast to prokaryotes where mRNA molecules exist for only a few minutes, the mRNA in eukaryotes is relatively stable. In mammalian cells, estimates of half-lifes of long-lived mRNAs vary from 24 to 100 hr, depending on the system (Singer and Penman, 1973). The rates of mRNA degradation can change dramatically in response to some biological or pharmacological stimuli, e.g. vitellogenin mRNA was
1
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DISCUSSION Actinomycin D did not interfere with resumption of pharyngeal pumping and worms became sensitive to SDS (Figs 2 and 3) during the early stages of recovery. Very high concentrations of the drug which completely prevented growth had little effect on pumping and although worms did swell slightly in diameter from intake of food, no longitudinal growth was observed. The failure of actinomycin D to affect dauer larva recovery indicates the mRNA synthesis is not required for exit from the dauer stage. However, failure of the worms to molt in the presence of high concentrations of the drug indicate that new gene
Fig. 5. Stained patterns of proteins from dauer larvae and dauers which had been in recovery medium for 12 hr in the presence and absence of actinomycin D. Proteins were separated on a 10% polyacrylamide gel and stained with Coomassie blue. 20/~g of protein extract was applied to each lane. Lane 1 (dauer), 2 (control), 3 (10#M act D), 4 (20 #M act D) and 5 (E. coli OP50, the food source used in the recovery medium).
Caenorhabditis dauer larva recovery--I
shown to be degraded with a half-life of 480 hr in oestrogen-treated cells compared to a half life of 16 hr in untreated cells (Brock and Shapiro, 1983). Similarly, histone m R N A is stable for months in Xenopus oocyctes but is degraded within hours upon fertilization (Ruderman et al., 1979; W o o d l a n d et al., 1979). Thus, our finding that m R N A synthesis seems not to be obligatory for dauer recovery suggests that the dauer larva may represent a developmental state analogous to unfertilized oocytes which store maternal m R N A s for long periods (reviewed by Davidson, 1986) or to diapausing insect embryos which contain a store of inactive m R N A s which are activated during post diapause development (see Saito et al., 1985, for example). Translation of stored m R N A may perhaps be triggered when dauer larvae perceive a food signal. If this is the case then protein synthesis inhibitors might be effective in preventing dauer recovery. In all accompanying paper (Reape and Burnell, 1991) we present the results obtained in an investigation of the effect of inhibitors of protein synthesis on dauer larva recovery. REFERENCES
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