J. Embryol. exp. Morph. Vol. 21, 1, pp. 55-70, February 1969 55

Printed in Great Britain

Nucleic acid and protein synthesis and patternregulation in hydra

II. Effect of inhibition of nucleic acid and protein synthesison hypostome formation

By S. G. CLARKSON1

From the Department of Biology as Applied to Medicine,Middlesex Hospital Medical School, London

In a previous paper (Clarkson, 1969) data were presented which indicate thathypostome determination is accompanied by a large and rapid burst of RNAsynthesis, a slight stimulation of protein synthesis, and no increase in DNAsynthesis. More direct evidence concerning the relative importance of thesemetabolic activities in hypostome determination is reported in this paper.

The experimental approach made use of the transplantation test of Webster& Wolpert (1966) in conjunction with some inhibitors of DNA, RNA andprotein synthesis, the rationale being that if these metabolic activities playimportant roles in the determination of the hypostome, then their inhibitionwould be expected to have severe effects on the time required for this process.

Regarding the inhibitors, hydroxyurea (HU) inhibits DNA synthesis in avariety of animal cells without altering rates of formation of RNA or protein(Young & Hodas, 1964; Yarbro, Kennedy & Barnum, 1965; Schwartz, Garo-falo, Sternberg & Philips, 1965). The suggestion has been made that its site ofaction is at the conversion of ribonucleotides to deoxyribonucleotides (Frenkel,Skinner & Smiley, 1964). Several modes of inhibition have been proposed for5-fluorouracil (FU), the principal ones being: (a) formation of thymidylic acid isinhibited by 5-fluorodeoxyuridylic acid, thereby blocking DNA synthesis(Heidelberger, Kaldor, Mukherjee & Danneberg, 1960); (b) abnormal RNA isproduced by the incorporation of FU in place of uracil, producing a toxicenzyme pattern (Gros et al. 1961); (c) nucleotide synthesis is inhibited, resultingin a reduced rate of RNA or DNA synthesis, or both (Skold, 1960). Actino-mycin D has been used as an effective and specific inhibitor of RNA synthesis ina number of systems. Its biological activity depends on its binding to DNA andthe consequent inhibition of DNA-dependent RNA synthesis by RNA poly-

1 Author's address: Department of Molecular, Cellular and Developmental Biology,University of Colorado, Boulder, Colorado 80302, U.S.A.

56 S. G. CLARKSON

merase (reviewed by Reich & Goldberg, 1964). Chloramphenicol inhibitsbacterial protein synthesis by specifically binding to the 50 S subunit of theribosomes (Vazquez, 1966), but its effects on protein synthesis in animal cells aremuch less clear.

MATERIALS AND METHODS

Hydra littoralis were used for all experiments. Details with regard to culturemethods, selection of animals, and transplantation experiments to investigatehypostomal properties are as given in Webster & Wolpert (1966).

The chemicals used in this study were obtained from the following sources:5-fluorouracil (FU) from Calbiochem, Los Angeles; hydroxyurea (HU), a giftfrom Squibbs and Sons Ltd., Twickenham; actinomycin D, a gift from Dr S. J.Coward, University of Georgia; and chloramphenicol (chloromycetin) fromParke, Davis and Co., Hounslow. They were dissolved in ' M ' solution and hydrawere treated in large volumes of the solutions at 26 °C. After treatment, whennecessary, the hydra were washed three times in 'M ' .

Details of biochemical procedures are as given in Clarkson (1969), the onlyexception being the determination of [3H]cytosine incorporation into DNA(Table 2). Since cytosine is also incorporated into RNA, the acid-insolubleradioactive samples were hydrolysed for 1 h at 37 °C in 0-3N-KOH. Afteraddition of cold 10% trichloroacetic acid, the precipitates were collected onMillipore filters and radioactivity determined as described previously (Clarkson,1968).

[3H]thymidine (5 c/mM), [3H]cytosine (100 mc/mM), [3H]uridine (3-33 c/mM),and [14C]algal protein hydrolysate (640 /*c/mg) were obtained from the Radio-chemical Centre, Amersham.

RESULTS

1. General effects of 5-fluorouracil, hydroxyurea, actinomycin Dand chloramphenicol

All four compounds delayed tentacle regeneration in animals cut at thesubhypostomal level, and treatment with FU at 100 /tg/ml, actinomycin D at10/ig/ml, or chloramphenicol at 600/^g/ml completely blocked tentacle budformation within 24 h. When treatment was continued beyond this time,tentacle buds were occasionally formed within 48 h of cutting but the majorityof animals disintegrated after 48-96 h without showing any signs of tentacleregeneration. Animals minus hypostome and tentacles treated for 24 h with HUat 1 mg/ml reconstituted tentacles bud within this time but required a further48 h to develop full tentacles when treatment was continued. Both types of effectshould be contrasted with untreated controls in which tentacles buds appearabout 18-20 h after cutting and full tentacles within at most a further 24 h.

Animals cut at the subhypostomal level treated for 24 h at the above con-centrations reconstituted full tentacles within 48-72 h of being washed and

Pattern regulation in hydra. II 57transferred to fresh 'M' . The tentacle pattern of such animals was usuallynormal, i.e. 5-7 equal-length tentacles arranged radially around the hypostome.Occasionally 3 or 4 tentacles were shorter than normal but usually theseincreased in length to fit the normal pattern.

None of these compounds caused any alterations of polarity. At non-toxicconcentrations reconstitution was exclusively monopolar in form, even inisolated digestive zones. At lethal doses animals were axiate with easily recogniz-able polarity right up to, and during, disintegration; invariably the last region todisintegrate was a bud at the medium stage of development.

These compounds severely reduced or, in the case of FU, completely preventedfurther bud initiation. In contrast, the main outgrowth and elongation of the budwas unaffected even by continuous treatments at the above concentrations, sothat initiated buds always developed the normal tubular appearance character-istic of later stages. This supports the earlier suggestion (Clarkson & Wolpert,1967) that bud elongation must be interpreted in terms of tissue movementrather than growth. In addition, tentacles were frequently observed on buds whiletentacle morphogenesis of the parent was completely inhibited during con-tinuous treatment with these compounds, an observation which suggests thatthere are distinct differences between the processes of regulation during buddingand regulation during regeneration.

To be reasonably certain that an effective inhibitor concentration had builtup by the time regeneration commenced, the compounds were added to intacthydra some considerable time before cutting in the isotope and transplantationexperiments. This pre-treatment period was 20 h in the case of FU, HU andchloramphenicol; 4h was chosen for actinomycin D in view of its toxicity.Animals remained viable for at least 20 h after cutting, but without showing anysigns of tentacle regeneration, following such pretreatment periods with thecompounds at the above concentrations.

The effects of 5-fluoro-2'-deoxyuridine (FUDR), cycloheximide, puromycinand colcemide on hydra were also investigated during the course of this work.FUDR at 500 /<g/ml had very similar biological effects to FU but was much lesseffective in reducing [3H]thymidine incorporation. Cycloheximide was extremelytoxic to hydra and 0-125 /*g/ml was the highest concentration that could beemployed. This concentration had no inhibitory effect on [14C]algal proteinhydrolysate incorporation, and no concentration was found to cause a delay indistal regeneration without concomitant toxicity. Puromycin at 20/^g/ml andcolcemide at 10/Ag/ml had similar biological effects to chloramphenicol, butneither compound produced the striking alterations of polarity described byWebster (1967) from his work on the effects of these compounds on hydra. Thereason for this is not clear at the moment although it should be noted that thehighest concentrations that could be employed in the present work were lowerthan those used by Webster. At the above concentrations, neither compoundsignificantly inhibited [14C]algal protein hydrolysate incorporation. Further

58 S. G. CLARKSON

details of the biological and biochemical effects of these compounds on hydraare given in Clarkson (1967); their effects on hypostome formation were notinvestigated because FU was found to be a more potent inhibitor of [3H]thy-midine incorporation than FUDR, and because the remaining three compoundshad no significant effect on [14C]algal protein hydrolysate incorporation.

2. Isotope experiments

(a) Effect of 5-fluorouracil on DNA, RNA and protein synthesis

Three batches of six intact hydra were treated with FU at lOO^g/ml in10~5M GSH in ' M' ; three batches of six control animals were similarly incubatedin GSH in ' M ' alone. After 20 h the animals were cut at the subhypostomal leveland the proximal parts incubated for 1 h in [3H]thymidine (5 c/misi) at 25 jnc/mlin 100 fig FU/ml in ' M \ Control animals were incubated in [3H]thymidine in' M ' alone. Identical experiments were performed with [3H]uridine (3-33 c/mM)at 25 //c/ml, and [14C]algal protein hydrolysate (640 juc/mg) at 20 /tc/ml. Resultsare shown in Table 1.

Table 1. Effect of 5-fluorouracil on the incorporation of [3H]thymidine intoDNA (A), [sH]uridine into RNA (B), and [uC]algal protein hydrolysate intoprotein (C)

FU-treated(c.p.m.//£g DNA,RNA or protein)

20-224-422-7

808-1

100

69160-465-8

Controls(c.p.m.//*g DNA,RNA or protein)

A156-5139-6165-4

B64-774-671-3

C85-477-672-1

% remainingDNA, RNA orprotein synthesis

12-917-513-7

Average 14-7 %

12-410-9140

Average 12-4%

80-977-891-3

Average 83-3 %

The results show that DNA synthesis, as measured by the incorporation of[3H]thymidine into DNA, is inhibited by 85 % during the first hour of distalregeneration following a 20 h pre-treatment with 100 fig FU/ml. Under identicalconditions RNA synthesis is also severely suppressed whereas protein synthesisis only slightly but significantly (P < 0-10) inhibited by this compound.

Pattern regulation in hydra. II 59

It should be noted that a reduction in [3H]thymidine incorporation would notbe predicted if inhibition of the methylation of deoxyuridylic acid to thymidylicacid were solely involved in the inhibition of DNA synthesis by FU, for thymi-dine is incorporated into the DNA pathway after this methylation step. How-ever, FU inhibits DNA synthesis in Ehrlich ascites tumour cells even whenthymidine is present (Lindner et al. 1963), a result which further suggests thatinhibition of thymidylic acid is not the only mechanism involved. Moreover, theincorporation of 25 /«c/ml [3H]cytosine (100 mc/mM) into DNA is also severelysuppressed during the first hour of distal regeneration following a 20 h pre-treatment with FU at 100/tg/ml. (Table 2).

Table 2. Effect of 5-fluorouracil on the incorporation of[zH]cytosine into DNA

FU-treated Controls % remaining(c.p.m.//*g DNA) (c.p.m.//*g DNA) DNA synthesis

13-9 42-5 32-7100 33-2 3018-5 29-8 28-5

Average 30-4 %

Table 3. Effect of hydroxyurea on the incorporation of [sH]thymidineinto DNA (A) and [zH]uridine into RNA (B)

HU-treated(c.p.m.//*g DNA

or RNA)

78-387-759-8

42154-640-3

Controls(c.p.m.//*g DNA

or RNA)

A

160-3207-21900

B

58-556-757-1

% remainingDNA or RNA

synthesis

48-842-331-5

Average 40-9 %

72096-370-6

Average 79-6%

(b) Effect of hydroxyurea on DNA and RNA synthesis

The incorporation of 25 /jc/ml [3H]thymidine (5 c/mM) into DNA, and of25/<c/ml [3H]uridine (3-33 c/mivi) into RNA were determined under the aboveconditions except that FU was replaced by HU at 1 mg/ml. Results are shown inTable 3.

The results demonstrate that DNA synthesis is inhibited by approximately60 % during the first hour of distal regeneration after a 20 h pre-treatment with

60 S. G. CLARKSON

HU at 1 mg/ml. Despite quite large variations between batches this inhibitionis very significant (P < 0-01) when the / test is applied. In contrast, the apparent20% inhibition of RNA synthesis is not significant (P > 0-10). It is thereforeconcluded that HU severely suppresses DNA synthesis under conditions thathave little effect on RNA synthesis.

(c) Effect of actinomycin D on RNA and protein synthesis

Three batches of six intact hydra were treated with actinomycin D at 10 /ig/mlin ' M ' ; three control batches were incubated in ' M' alone. After 4 h the animalswere cut at the subhypostomal level and the proximal parts incubated for 1 h in3H-uridine (3-33 c/mM) at 50/tc/ml in actinomycin D at 10/tg/ml; the controls

Table 4. Effect of actinomycin D on the incorporation of [zH]uridineinto RNA (A) and [uC]algal protein hydrolysate into protein (B)

ActinomycinD-treated

(c.p.m.//*g RNAor protein)

20-919-423-8

38-539-538-4

Controls(c.p.m.//*g RNA

or protein)

A

166-71190150-4

B50-456-144-4

% remainingRNA or protein

synthesis

12-516-315-8

Average 14-9 %

76-470-486-5

Average 77-8%

Table 5. Effect of chloramphenicol on the incorporation of[uC]algalprotein hydrolysate into protein

Chloramphenicol-treated Controls % remaining

(c.p.m.//*g protein) (c.p.m.//*g protein) protein synthesis

19-216-619-7

83-571-482-9

23 023-223-8

Average 23-3%

were labelled with [3H]uridine in ' M ' alone. An identical experiment wasperformed with [14C]algal protein hydrolysate (640//c/mg) at 15 jLtc/ml. Resultsare shown in Table 4; it can be seen that actinomycin D inhibits RNA synthesisto a level less than 15 % of the control level under conditions that have only aslight inhibitory effect on protein synthesis.

Pattern regulation in hydra. II 61

(d) Effect of chloramphenicol on protein synthesis

The procedure in this experiment was similar to that in the last except thatactinomycin D was replaced by chloramphenicol at 600 /tg/ml and that a 20 hpre-treatment period was employed. The specific activities of the control andchloramphenicol-treated hydra following a 1 h pulse with 20 /^c/ml [14C]algalprotein hydrolysate (640 /^c/mg) are shown in Table 5. The results demonstratethat protein synthesis is severely inhibited during the first hour of distal re-generation after a 20 h pre-treatment with 600 jitg chloramphenicol/ml.

3. Transplantation experiments

(a) Effect of 5-fluorouracil on hypostome formation

The effect of FU on the formation of a new hypostome was investigated usingthe transplantation test of Webster & Wolpert (1966). Intact hydra were treatedfor 20 h with FU at 100 ̂ g/ml in 10"5M GSH in 'M ' , cut at the subhypostomallevel, and the proximal parts left to reconstitute in fresh FU medium. At various

Table 6. Effect of 5-fluorouracil on hypostome formationfrom the subhypostomal region

Source of graft

Control subhypostome

FU-treatedsubhypostome

Time aftercutting whengrafted (h)

<H46

o-i-6916

No. ofsuccessful

grafts

172021

17201919

No. of animalswith secondary

axis

09(45%)

16(76%)01(5%)5(26%)

12(63%)

times after cutting, the distal tip of the reconstituting piece was removed andtransplanted to the mid-digestive zone of an untreated control host. All graftingoperations were performed in ' M ' alone and the animals were kept in ' M ' forthe duration of the experiment. The results of control and FU-treated sub-hypostomal grafts are shown in Table 6.

The results demonstrate a distinct difference between the control and FU-treated subhypostomes in the time at which a determined hypostome is formed.Following Webster & Wolpert (1966), a comparison of the times required forhypostome determination in the two situations is possible if an estimate is madeof the time for 50 % of the pieces to become determined (T50). For the controlsubhypostomal region T50 = 4£h; for the FU-treated subhypostomal regionT50 = 13|h. The secondary axes were type 1 or 3 inductions (Webster &Wolpert, 1966) from both the control and FU-treated subhypostomes.

62 S. G. CLARKSON

(b) Effect of hydroxyurea on hypostome formation

The procedure in this experiment was the same as that in the last except thathydra were treated with HU at 1 mg/ml instead of FU. The number of animalsproducing induced secondary axes as a result of transplantation of the distal tipsof reconstituting control and HU-treated hydra is shown in Table 7.

Table 7. Effect of hydroxyurea on hypostome formationfrom the subhypostomal region

Time after No. of No. of animalscutting when successful with secondary

Source of graft grafted (h) grafts axis

Control subhypostome 6 20 15 (75 %)HU-treated 0 - | 14 0subhypostome 5 18 8(44%)

8 18 14(78%)

Table 7 shows that the majority of the pieces from control subhypostomesinduce secondary axes 6 h after cutting at the subhypostomal level, thusconfirming the findings of the previous experiment and those of Webster &Wolpert (1966). In contrast to FU, HU treatment does not lead to a very greatdelay in the time required for hypostome formation. For the HU-treatedsubhypostomal region T50 = 5^h; all the positive cases were type 1 or 3inductions. Similarly, the delay between hypostome determination and tentacleregeneration was not as great as in the case of FU-treated animals. After a 20 hpre-treatment, HU-treated hydra developed tentacle buds within 24 h of cuttingat the subhypostomal level. This delay of approximately 19 h between hypostomeformation and tentacle morphogenesis is similar to that found in untreatedcontrols (13-15 h), and should be contrasted with that seen in FU-treated hydra(> 48 h).

(c) Effect of actinomycin D on hypostome formation

After a 4 h treatment with actinomycin D at 10 /*g/ml, hydra were cut at thesubhypostomal level, placed in fresh actinomycin medium, and at various timesthereafter the distal tip of a reconstituting animal was transplanted to thedigestive zone of an untreated host hydra. Results are shown in Table 8.

Actinomycin D treatment caused quite a considerable delay in the timerequired for the grafts to become determined: T50 = 10 h. The term 'hypostomedetermination' has not been applied to these results because, in striking contrastto the behaviour of control subhypostomal grafts, the great majority of secon-dary axes induced after actinomycin treatment were in the form of a typicalpeduncle and basal disk. Thus twenty-two of the twenty-four secondary axesinduced after actinomycin treatment were of this type, and only one type 1

Pattern regulation in hydra. II 63induction was obtained as a result of grafting at 6 h after cutting and anothertype 1 induction at 16 h. After a 4h pre-treatment, animals continuouslytreated with actinomycin D at 10 /*g/ml disintegrated within 24-36 h of cuttingat the subhypostomal level without showing any signs of tentacle regeneration.In view of the type of secondary axes induced by grafts from actinomycin D-treated animals, it is also worth noting that these animals showed no signs of theformation of proximal regions at the distal end.

Table 8. Effect of actinomycin D on hypos tome formationfrom the subhypostomal region

Source of graft

Control subhypostome

Actinomycin D-treatedsubhypostome

Time aftercutting whengrafted (h)

6

0-4616

Table 9. Effect of chloramphenicol onfrom the subhypostomal

Source of graft

Time aftercutting whengrafted (h)

No. ofsuccessful

grafts

19

142122

No. of animalswith secondary

axis

14(74%)

07(33%)

17(77%)

hypostome formationregion

No. ofsuccessful

grafts

No. of animalswith secondary

axis

Control subhypostome 6 15 12(80%)

Chloramphenicol-treated 0-i 12 0subhypostome 6 20 0

16 18 1 (6%)24 19 4 (21 %)

(d) Effect of chloramphenicol on hypostome formation

The procedure in this experiment was the same as that in the last exceptactinomycin D was replaced by chloramphenicol at 600 /*g/ml and that a 20 hpre-treatment was employed. The number of animals producing secondary axesafter transplantation of control and chloramphenicol-treated subhypostomes isshown in Table 9.

No T50 was obtained for the chloramphenicol-treated subhypostomesbecause even after 24 h only a small number of transplanted pieces induced. Thesecondary axes that were obtained were all type 1 or 3 inductions. The experi-ment was not continued beyond 24 h for the animals died within 24-36 h ofcutting at the subhypostomal level during chloramphenicol treatment, disinte-gration invariably commencing at the distal end. This experiment shows there-fore that chloramphenicol treatment causes a very considerable delay in the time

64 S. G. CLARKSON

required for hypostome determination from the subhypostomal region. Whileno T50 could be obtained for chloramphenicol-treated subhypostomes, it isevident that this delay is at least 20 h.

DISCUSSION

Some consideration must be given to the inhibition of nucleic acid and proteinsynthesis by the four compounds during the time required for the grafts tobecome determined in the transplantation experiments. After a 4 or 20 h pre-treatment period, continuous treatment of regenerating animals with thecompounds at the above concentrations results in their disintegration within24-48 h of cutting at the subhypostomal level. It is considered extremelyunlikely, therefore, that nucleic acid and protein synthesis would recoverbeyond the first hour of distal regeneration under these conditions, and it isassumed that during the time required for the grafts to become determined in thetransplantation experiments the extent of inhibition of DNA, RNA, or proteinsynthesis in the treated samples is at least the same as that recorded within thefirst hour of cutting at the subhypostomal level. This has been shown to be thecase for the inhibition of [3H]thymidine incorporation by HU (Clarkson, 1967).

A second point that requires some comment is the possibility that DNA, RNA,or protein synthesis in the grafted pieces could recover following their trans-plantation to untreated host hydra. Since the pieces were transplanted to thesame region of the host, the formation of a secondary axis as a result of trans-plantation depends upon changes in the transplanted piece, specifically theacquisition of inductive ability and resistance to absorption (Webster & Wolpert,1966). The nature of the test implies, however, that these properties are acquiredbefore transplantation is performed, i.e. while the prospective grafts are stillbeing treated with one of the inhibitors. It is suggested therefore that the possiblerecovery of DNA, RNA or protein synthesis in the grafted pieces cannot accountfor the formation of secondary axes in the transplantation experiments.

The finding that HU severely inhibits the incorporation of [3H]thymidine intoDNA in regenerating hydra while having little effect on [3H]uridine incorpora-tion into RNA is in accord with the work on the effects of this compound onHeLa cells (Young & Hodas, 1964), ascites tumour cells (Yarbro et al. 1965),and regenerating rat liver (Schwartz et al. 1965). Under conditions of HU treat-ment that cause a 60 % reduction in [3H]thymidine incorporation (Table 3), thetime required for hypostome determination from the subhypostomal region isincreased by only 22 % (4£to 5\ h, Table 7). It is therefore concluded that DNAsynthesis plays no major role in the acquisition of organizing properties by thehypostome.

At first sight the data on the effect of FU on distal regeneration appear torefute this conclusion. Thus, FU causes a threefold increase in the time requiredfor hypostome determination (4^ to 13^ h, Table 6) and, on the basis of the

Pattern regulation in hydra. II 65

inhibition of [3H]thymidine and [3H]cytosine incorporation (Tables 1, 2), it isassumed that DNA synthesis is inhibited by at least 70% during this period.However, the distinct difference between HU and FU in their effect on the timefor hypostome formation is unlikely to be due solely to their different levels ofinhibition of DNA synthesis. Rather, it seems more reasonable to suggest thatthe T50 of 13£h for FU-treated subhypostomes, when compared to the 5^hT50 of HU-treated subhypostomes, reflects the fact that [3H]uridine incorpora-tion into RNA is severely suppressed by FU (Table 1), but not by HU (Table 3)It is also possible that this may be due in part to a differential effect on proteinsynthesis, for FU slightly inhibits protein synthesis in addition to, or as aconsequence of, its effect on RNA synthesis (Table 1). It is therefore concludedthat the delay of hypostome formation in FU-treated hydra is not a consequenceof the inhibition of DNA synthesis, but rather of the inhibition of RNA andpossibly also protein synthesis. This is consistent with the earlier suggestion(Clarkson, 1969) that RNA and protein synthesis play important roles in thedetermination of a hypostome.

Actinomycin D inhibits RNA synthesis by 85 % during the first hour of distalregeneration following a 4 h pre-treatment (Table 4) and it is assumed that thislevel of inhibition is at least maintained during the time required for the actino-mycin D-treated grafts to become determined in the transplantation experimentsshown in Table 8. The customary action of actinomycin is to inhibit DNA-primedRNA synthesis, and the nature of the residual 15% actinomycin-resistantfraction is therefore important for the interpretation of the effects of thiscompound at a biochemical level. If this 15 % incorporation reflects even a smallamount of synthesis of an active messenger RNA fraction, it could account for agreat deal of protein synthesis and perhaps have important developmentaleffects. On the other hand, there are at least two other possible explanations forthis apparent synthesis of RNA in the presence of actinomycin D: (1) some[3H]uridine was incorporated into DNA via conversion to thymine; and (2) some[3H]uridine was incorporated into the terminal CCA sequence of transfer RNAafter conversion of uracil to cytosine (Franklin, 1963).

While it remains to be demonstrated that actinomycin D inhibits the synthesisof RNA molecules large enough to be thought of as normal messenger RNA inhydra, the finding that the incorporation of [14C]algal protein hydrolysate intoprotein is inhibited by only 23 % after RNA synthesis has been substantiallyinhibited (Table 4) strongly suggests that relatively stable messenger RNAsexist in hydra.

At a biological level the effect of actinomycin D is extremely interesting. Thetransplantation experiments indicate that hypostome formation is severelyinhibited by actinomycin, at least as judged by the small number of type 1induced secondary axes. On the other hand, resistance to absorption wasacquired within a T 50 of 10 h, the great majority of the induced axes being in theform of a peduncle and basal disk (Table 8). This suggests very strongly that

66 S. G. CLARKSON

inductive ability and resistance to absorption are quite distinct properties. It isinteresting to note that a dissociation of these two properties has been suggestedby Webster (1967) from a quite different approach. If the resistance to absorp-tion normally displayed by the determined hypostome is dependent on resistanceto inhibition and therefore upon high threshold (Webster, 1967), the fact thatresistance to absorption can be acquired in the presence of actinomycin Dsuggests that either the 15% actinomycin-resistant RNA fraction is actuallyall the template material necessary for the acquisition of high threshold, theinhibited fraction being somehow irrelevant, or threshold is predominantlyunder translation control. It is also possible, although less likely, that this factoris independent of all RNA synthesis. Conversely, the inability of actinomycinD-treated subhypostomes to induce type 1 secondary axes following trans-plantation might suggest that the normal acquisition of inductive ability by thehypostome involves dramatic changes in transcription.

An alternative explanation for the secondary axes induced by actinomycinD-treated subhypostomes is possible if it is assumed that they reflect thedetermination of a basal disk at the distal end. It is known that a piece of basaldisk retains its identity when transplanted (Burt, 1925; Burnett, 1959), althoughit has been suggested that the induced basal disks are made entirely from thegraft tissue (Burnett, 1961), unlike the induction of a secondary axis by ahypostome which utilizes host tissue (Yao, 1945). Whether or not host materialis incorporated into the induced basal disk, it seems clear that this regionpossesses the ability to resist absorption. In addition, a basal disk can form atthe distal end of regenerating hydra under some circumstances, e.g. treatmentwith Cleland's reagent (S. G. Clarkson, unpublished observations). Whether asimilar situation exists in the case of actinomycin treatment is difficult todetermine for animals kept in actinomycin disintegrated within 24-36 h ofcutting at the subhypostomal level without showing signs of either distal orproximal structures at the distal end. It should be noted, however, that basal diskformation from the proximal end of hydra reconstituting in ' M ' requiresabout 20 h (Webster, 1964). It is therefore difficult to envisage how a basal diskcould form within such a short time as 10 h at the distal end of the axis. More-over, the secondary axes obtained in the present study were significantly largerthan the size of the implanted pieces. This suggests that some reorientation ofhost material did occur and, therefore, that the induced axes reflect the resistanceto absorption of the hypostome rather than the determination of a basal disk atthe distal end.

It will be evident that certain difficulties arise when the effects of FU andactinomycin D on RNA synthesis and hypostome determination are compared.It was suggested earlier that FU causes a threefold increase in the time requiredfor hypostome determination by virtue of its inhibition of RNA rather thanDNA synthesis. However, at very similar levels of inhibition of RNA synthesis,actinomycin D-treated subhypostomes appear to acquire only resistance to

Pattern regulation in hydra. II 67absorption whereas FU-treated subhypostomes acquire both resistance toabsorption and inductive ability. The results are therefore difficult to correlatewhen put in terms of total RNA synthesis. In addition, there exists no completelysatisfactory explanation of the inhibition of RNA synthesis by FU, althoughHeidelberger (1965) has made the interesting proposal that FU incorporatedinto messenger RNA does not affect the process of translation whereas FU doesappear to have an inhibitory effect on transcription, possibly through itsappearance at sites on messenger RNA normally occupied by cytosine. Thefinding that [14C]algal protein hydrolysate incorporation is inhibited by only 17 %by FU (Table 1) is consistent with this hypothesis, or it might be interpreted asmeaning that a large proportion of protein synthesis during early distal regenera-tion is coded for by messengers of relatively long half-lives. In spite of ignoranceof the detailed biochemical effects of FU it would thus appear that the delay ofdistal regeneration in FU-treated subhypostomes is due primarily to inhibitionof RNA synthesis.

On the other hand, chloramphenicol severely inhibits [14C]algal protein hydro-lysate incorporation into protein (Table 5) and under these conditions hypostomedetermination is delayed by at least 20 h (Table 9). This suggests that proteinsynthesis plays a major role in the acquisition of resistance to absorption andinductive ability by the hypostome, although the results may be due more simplyto the toxicity of chloramphenicol to hydra. While inhibition of bacterial proteinsynthesis by this compound has been amply documented, conflicting observationshave been made on its effects on protein synthesis in other systems. As with FU,it is clear that further work will be necessary before a biochemical explanationis possible of the delay of hypostome determination by chloramphenicol.

In spite of the inherent limitations of this level of analysis, the results areconsistent with the earlier suggestion (Clarkson, 1969) that RNA and proteinsynthesis play important roles in the control of polarized regulation in hydra,and with the recent evidence which suggests that growth plays no major role inthis process (Campbell, 1967; Clarkson & Wolpert, 1967; Webster, 1967;Clarkson, 1969). The experiments with actinomycin D in particular suggest thatan important proportion of protein synthesis in hydra is performed utilizingrelatively stable RNA templates. Thus, threshold might be predominantly undertranslational control and this perhaps could account for the assumed stability ofthis factor (Webster, 1966a, b). This is also consistent with the view that thestimulus for reconstitution is not 'activation', but release from inhibition (Rose& Rose, 1941; Webster, 1966«). Conversely, the synthesis of RNA moleculeshaving only a limited half-life could provide the means of control for a transitoryresponse such as tentacle morphogenesis. These suggestions must be regarded asextremely tentative, however, for it has yet to be demonstrated that proteinsynthesis in hydra is of the sort that requires template RNA, i.e. polysomal, orindeed that the sedimentation profiles of hydra RNA are similar to those ofother animal cells.

5-2

68 S. G. CLARKSON

SUMMARY

1. The effects of inhibition of nucleic acid and protein synthesis on the timerequired for hypostome formation from the subhypostomal region wereinvestigated using biochemical and transplantation techniques.

2. Hydroxyurea can cause a 22 % increase in the time required for hypostomeformation. The incorporation of [3H]thymidine into DNA is severely inhibitedunder these conditions, whereas RNA synthesis appears to be essentially normal.

3. 5-fluorouracil severely inhibits the incorporation of both [3H]thymidineand [3H]cytosine into DNA, and the incorporation of [3H]uridine into RNAunder conditions that apparently produce only a slight inhibition of proteinsynthesis. The time for hypostome determination is increased threefold underthese conditions. This delay is considered to be due primarily to the inhibition ofRNA synthesis.

4. Actinomycin D substantially suppresses [3H]uridine incorporation intoRNA but has only a slight inhibitory effect on [14C]algal protein hydrolysateincorporation into protein. Hypostome formation is not completely inhibitedunder these conditions for actinomycin D-treated subhypostomes can acquireresistance to absorption. The majority of the hypostomes formed, however, donot possess normal inductive ability. This suggests that the factors responsiblefor the inductive ability of the hypostome (i.e. tentacle morphogenesis) aredistinct from those controlling resistance to absorption. This might reflectdifferences in the stability of messenger RNAs.

5. Chloramphenicol severely inhibits [14C]algal protein hydrolysate in-corporation into protein. The time for hypostome determination appears to bedelayed by at least 20 h under these conditions, but this may reflect the toxicityof chloramphenicol to hydra.

6. The significance of the results is discussed in relation to concepts developedby other workers to explain polarized regulation in hydra.

RESUME

La synthese des acides nucleiques et des proteines et la regulation de la morpho-genese chez Vhydre. II. Les ejfets de Vinhibition de la synthese des acidesnucleiques et des proteines sur la formation de Vhypostome

1. Des techniques biochimiques et des transplantations ont ete utilisees pouretudier les effets de l'inhibition de la synthese des acides nucleiques et desproteines sur la duree de formation de la region orale (hypostome) de l'hydre, apartir de la region sub-orale.

2. La duree de formation de l'hypostome est augmentee de 22% par untraitement a l'hydroxyuree: l'incorporation de [3H]thymidine dans le DNA estnettement inhibee dans ces conditions, alors que la synthese du RNA resterelativement normale.

Pattern regulation in hydra. II 693. L'incorporation de 3H-thymidine et de 3H-cytosine dans le DNA, de

meme que celle de la 3H-uridine dans le RNA, est tres fortement reduite enpresence de 5-fluorouracil, alors que la reduction de la synthese proteique n'estque partielle. Le temps de formation de l'hypostome est triple dans cesconditions.

4. L'actinomycine reduit tres fortement l'incorporation de 3H-uridine dans leRNA, mais n'a qu'un tres leger effet inhibiteur sur l'incorporation d'un hydro-lysat de proteines-C14. L'inhibition de la formation de l'hypostome n'estcependant pas complete, puisqu'apres un traitement a l'actinomycine, uneresistance a l'absorption de cet antibiotique se developpe. La plupart deshypostomes formes dans de telles conditions experimentales ne possedentcependant pas une capacite inductrice normale. Les facteurs responsables dupouvoir inducteur et controlant la morphogenese des tentacules seraient done denature differente de ceux influencant la resistance a l'absorption. II est possibleque ce soit le reflet de differences de stabilite au niveau des mRNAs.

5. Le chloramphenicol inhibe nettement l'incorporation d'hydrolysat deproteines-C14 dans les proteines. Un retard de 20 h s'observe dans le develop-pement de l'hypostome en presence de cet antibiotique, mais il n'est pas excluque cet effet soit du a la toxicite du produit.

6. La signification de ces resultats est discutee par rapport aux differenteshypotheses deja emises par d'autres auteurs pour expliquer la regulation de lamorphogenese chez l'hydre.

I am deeply indebted to Professor Lewis Wolpert for his advice and encouragement. I amgrateful to Squibbs and Sons Ltd. for a gift of hydroxyurea, and Dr S. J. Coward for a gift ofactinomycin D. We wish to thank the Agricultural Research Council for a scintillationcounter, and the Nuffield Foundation for their support of this work.

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(Manuscript received 30 April 1968)