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J. Embryol. exp. Morph. Vol. 21, 1, pp. 55-70, February 1969 Printed in Great Britain 55 Nucleic acid and protein synthesis and pattern regulation in hydra II. Effect of inhibition of nucleic acid and protein synthesis on hypostome formation By S. G. CLARKSON 1 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 that hypostome determination is accompanied by a large and rapid burst of RNA synthesis, a slight stimulation of protein synthesis, and no increase in DNA synthesis. More direct evidence concerning the relative importance of these metabolic 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 and protein synthesis, the rationale being that if these metabolic activities play important roles in the determination of the hypostome, then their inhibition would be expected to have severe effects on the time required for this process. Regarding the inhibitors, hydroxyurea (HU) inhibits DNA synthesis in a variety of animal cells without altering rates of formation of RNA or protein (Young & Hodas, 1964; Yarbro, Kennedy & Barnum, 1965; Schwartz, Garofalo, Sternberg & Philips, 1965). The suggestion has been made that its site of action is at the conversion of ribonucleotides to deoxyribonucleotides (Frenkel, Skinner & Smiley, 1964). Several modes of inhibition have been proposed for 5-fluorouracil (FU), the principal ones being: (a) formation of thymidylic acid is inhibited by 5-fluorodeoxyuridylic acid, thereby blocking DNA synthesis (Heidelberger, Kaldor, Mukherjee & Danneberg, 1960); (b) abnormal RNA is produced by the incorporation of FU in place of uracil, producing a toxic enzyme pattern (Gros et al. 1961); (c) nucleotide synthesis is inhibited, resulting in a reduced rate of RNA or DNA synthesis, or both (Skold, 1960). Actinomycin D has been used as an effective and specific inhibitor of RNA synthesis in a number of systems. Its biological activity depends on its binding to DNA and the consequent inhibition of DNA-dependent RNA synthesis by RNA poly1 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 inhibits bacterial protein synthesis by specifically binding to the 50 S subunit of the ribosomes (Vazquez, 1966), but its effects on protein synthesis in animal cells are much less clear. MATERIALS AND METHODS Hydra littoralis were used for all experiments. Details with regard to culture methods, selection of animals, and transplantation experiments to investigate hypostomal 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 gift from Squibbs and Sons Ltd., Twickenham; actinomycin D, a gift from Dr S. J. Coward, University of Georgia; and chloramphenicol (chloromycetin) from Parke, Davis and Co., Hounslow. They were dissolved in' M ' solution and hydra were treated in large volumes of the solutions at 26 °C. After treatment, when necessary, the hydra were washed three times in ' M ' . Details of biochemical procedures are as given in Clarkson (1969), the only exception being the determination of [3H]cytosine incorporation into DNA (Table 2). Since cytosine is also incorporated into RNA, the acid-insoluble radioactive samples were hydrolysed for 1 h at 37 °C in 0-3N-KOH. After addition of cold 10% trichloroacetic acid, the precipitates were collected on Millipore 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 Radiochemical Centre, Amersham. RESULTS 1. General effects of 5-fluorouracil, hydroxyurea, actinomycin D and chloramphenicol All four compounds delayed tentacle regeneration in animals cut at the subhypostomal level, and treatment with FU at 100 /tg/ml, actinomycin D at 10/ig/ml, or chloramphenicol at 600/^g/ml completely blocked tentacle bud formation within 24 h. When treatment was continued beyond this time, tentacle buds were occasionally formed within 48 h of cutting but the majority of animals disintegrated after 48-96 h without showing any signs of tentacle regeneration. Animals minus hypostome and tentacles treated for 24 h with HU at 1 mg/ml reconstituted tentacles bud within this time but required a further 48 h to develop full tentacles when treatment was continued. Both types of effect should be contrasted with untreated controls in which tentacles buds appear about 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 concentrations reconstituted full tentacles within 48-72 h of being washed and Pattern regulation in hydra. II 57 transferred to fresh ' M ' . The tentacle pattern of such animals was usually normal, i.e. 5-7 equal-length tentacles arranged radially around the hypostome. Occasionally 3 or 4 tentacles were shorter than normal but usually these increased in length to fit the normal pattern. None of these compounds caused any alterations of polarity. At non-toxic concentrations reconstitution was exclusively monopolar in form, even in isolated digestive zones. At lethal doses animals were axiate with easily recognizable polarity right up to, and during, disintegration; invariably the last region to disintegrate was a bud at the medium stage of development. These compounds severely reduced or, in the case of FU, completely prevented further bud initiation. In contrast, the main outgrowth and elongation of the bud was unaffected even by continuous treatments at the above concentrations, so that initiated buds always developed the normal tubular appearance characteristic of later stages. This supports the earlier suggestion (Clarkson & Wolpert, 1967) that bud elongation must be interpreted in terms of tissue movement rather than growth. In addition, tentacles were frequently observed on buds while tentacle morphogenesis of the parent was completely inhibited during continuous treatment with these compounds, an observation which suggests that there are distinct differences between the processes of regulation during budding and regulation during regeneration. To be reasonably certain that an effective inhibitor concentration had built up by the time regeneration commenced, the compounds were added to intact hydra some considerable time before cutting in the isotope and transplantation experiments. This pre-treatment period was 20 h in the case of FU, HU and chloramphenicol; 4 h was chosen for actinomycin D in view of its toxicity. Animals remained viable for at least 20 h after cutting, but without showing any signs of tentacle regeneration, following such pretreatment periods with the compounds at the above concentrations. The effects of 5-fluoro-2'-deoxyuridine (FUDR), cycloheximide, puromycin and 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 less effective in reducing [3H]thymidine incorporation. Cycloheximide was extremely toxic to hydra and 0-125 /*g/ml was the highest concentration that could be employed. This concentration had no inhibitory effect on [14C]algal protein hydrolysate incorporation, and no concentration was found to cause a delay in distal regeneration without concomitant toxicity. Puromycin at 20/^g/ml and colcemide at 10/Ag/ml had similar biological effects to chloramphenicol, but neither compound produced the striking alterations of polarity described by Webster (1967) from his work on the effects of these compounds on hydra. The reason for this is not clear at the moment although it should be noted that the highest concentrations that could be employed in the present work were lower than those used by Webster. At the above concentrations, neither compound significantly inhibited [14C]algal protein hydrolysate incorporation. Further 58 S. G. CLARKSON details of the biological and biochemical effects of these compounds on hydra are given in Clarkson (1967); their effects on hypostome formation were not investigated because FU was found to be a more potent inhibitor of [3H]thymidine incorporation than FUDR, and because the remaining three compounds had 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 in 10~ 5 M GSH in' M ' ; three batches of six control animals were similarly incubated in GSH in ' M ' alone. After 20 h the animals were cut at the subhypostomal level and the proximal parts incubated for 1 h in [3H]thymidine (5 c/misi) at 25 jnc/ml in 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. Results are shown in Table 1. Table 1. Effect of 5-fluorouracil on the incorporation of [3H]thymidine into DNA (A), [sH]uridine into RNA (B), and [uC]algal protein hydrolysate into protein (C) FU-treated (c.p.m.//£g DNA, RNA or protein) Controls (c.p.m.//*g DNA, RNA or protein) 20-2 24-4 22-7 A 156-5 139-6 165-4 80 8-1 100 B 64-7 74-6 71-3 691 60-4 65-8 C 85-4 77-6 72-1 % remaining DNA, RNA or protein synthesis 12-9 17-5 13-7 Average 14-7 % 12-4 10-9 140 Average 12-4% 80-9 77-8 91-3 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 distal regeneration following a 20 h pre-treatment with 100 fig FU/ml. Under identical conditions RNA synthesis is also severely suppressed whereas protein synthesis is only slightly but significantly (P < 0-10) inhibited by this compound. Pattern regulation in hydra. II 59 3 It should be noted that a reduction in [ H]thymidine incorporation would not be predicted if inhibition of the methylation of deoxyuridylic acid to thymidylic acid were solely involved in the inhibition of DNA synthesis by FU, for thymidine is incorporated into the DNA pathway after this methylation step. However, FU inhibits DNA synthesis in Ehrlich ascites tumour cells even when thymidine is present (Lindner et al. 1963), a result which further suggests that inhibition of thymidylic acid is not the only mechanism involved. Moreover, the incorporation of 25 /«c/ml [3H]cytosine (100 mc/mM) into DNA is also severely suppressed during the first hour of distal regeneration following a 20 h pretreatment with FU at 100/tg/ml. (Table 2). Table 2. Effect of 5-fluorouracil on the incorporation of [zH]cytosine into DNA FU-treated (c.p.m.//*g DNA) Controls (c.p.m.//*g DNA) 13-9 100 8-5 42-5 33-2 29-8 % remaining DNA synthesis 32-7 301 28-5 Average 30-4 % Table 3. Effect of hydroxyurea on the incorporation of [sH]thymidine into DNA (A) and [zH]uridine into RNA (B) HU-treated (c.p.m.//*g DNA or RNA) Controls (c.p.m.//*g DNA or RNA) 78-3 87-7 59-8 A 160-3 207-2 1900 421 54-6 40-3 B 58-5 56-7 57-1 % remaining DNA or RNA synthesis 48-8 42-3 31-5 Average 40-9 % 720 96-3 70-6 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 of 25/<c/ml [3H]uridine (3-33 c/mivi) into RNA were determined under the above conditions except that FU was replaced by HU at 1 mg/ml. Results are shown in Table 3. The results demonstrate that DNA synthesis is inhibited by approximately 60 % 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 inhibition is very significant (P < 0-01) when the / test is applied. In contrast, the apparent 20% inhibition of RNA synthesis is not significant (P > 0-10). It is therefore concluded that HU severely suppresses DNA synthesis under conditions that have 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/ml in ' M ' ; three control batches were incubated in ' M ' alone. After 4 h the animals were cut at the subhypostomal level and the proximal parts incubated for 1 h in 3 H-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]uridine into RNA (A) and [uC]algal protein hydrolysate into protein (B) Actinomycin D-treated (c.p.m.//*g RNA or protein) Controls (c.p.m.//*g RNA or protein) 20-9 19-4 23-8 A 166-7 1190 150-4 38-5 39-5 38-4 B 50-4 56-1 44-4 % remaining RNA or protein synthesis 12-5 16-3 15-8 Average 14-9 % 76-4 70-4 86-5 Average 77-8% Table 5. Effect of chloramphenicol on the incorporation of[uC]algal protein hydrolysate into protein Chloramphenicoltreated (c.p.m.//*g protein) Controls (c.p.m.//*g protein) 19-2 16-6 19-7 83-5 71-4 82-9 % remaining protein synthesis 23 0 23-2 23-8 Average 23-3% were labelled with [3H]uridine in ' M ' alone. An identical experiment was performed with [14C]algal protein hydrolysate (640//c/mg) at 15 jLtc/ml. Results are shown in Table 4; it can be seen that actinomycin D inhibits RNA synthesis to a level less than 15 % of the control level under conditions that have only a slight inhibitory effect on protein synthesis. 61 Pattern regulation in hydra. II (d) Effect of chloramphenicol on protein synthesis The procedure in this experiment was similar to that in the last except that actinomycin D was replaced by chloramphenicol at 600 /tg/ml and that a 20 h pre-treatment period was employed. The specific activities of the control and chloramphenicol-treated hydra following a 1 h pulse with 20 /^c/ml [14C]algal protein hydrolysate (640 /^c/mg) are shown in Table 5. The results demonstrate that protein synthesis is severely inhibited during the first hour of distal regeneration 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 using the transplantation test of Webster & Wolpert (1966). Intact hydra were treated for 20 h with FU at 100 ^g/ml in 10" 5 M GSH in ' M ' , cut at the subhypostomal level, and the proximal parts left to reconstitute in fresh FU medium. At various Table 6. Effect of 5-fluorouracil on hypostome formation from the subhypostomal region Source of graft Control subhypostome Time after cutting when grafted (h) No. of successful grafts <H 17 20 21 o-i- 17 20 19 19 4 6 FU-treated subhypostome 6 9 16 No. of animals with secondary axis 0 9(45%) 16(76%) 0 1(5%) 5(26%) 12(63%) times after cutting, the distal tip of the reconstituting piece was removed and transplanted to the mid-digestive zone of an untreated control host. All grafting operations were performed in ' M ' alone and the animals were kept in ' M ' for the duration of the experiment. The results of control and FU-treated subhypostomal grafts are shown in Table 6. The results demonstrate a distinct difference between the control and FUtreated subhypostomes in the time at which a determined hypostome is formed. Following Webster & Wolpert (1966), a comparison of the times required for hypostome determination in the two situations is possible if an estimate is made of the time for 50 % of the pieces to become determined (T50). For the control subhypostomal region T50 = 4£h; for the FU-treated subhypostomal region T50 = 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 that hydra were treated with HU at 1 mg/ml instead of FU. The number of animals producing induced secondary axes as a result of transplantation of the distal tips of reconstituting control and HU-treated hydra is shown in Table 7. Table 7. Effect of hydroxyurea on hypostome formation from the subhypostomal region Source of graft Control subhypostome HU-treated subhypostome Time after cutting when grafted (h) No. of successful grafts No. of animals with secondary axis 6 0-| 5 8 20 14 18 18 15 (75 %) 0 8(44%) 14(78%) Table 7 shows that the majority of the pieces from control subhypostomes induce secondary axes 6 h after cutting at the subhypostomal level, thus confirming the findings of the previous experiment and those of Webster & Wolpert (1966). In contrast to FU, HU treatment does not lead to a very great delay in the time required for hypostome formation. For the HU-treated subhypostomal region T50 = 5^h; all the positive cases were type 1 or 3 inductions. Similarly, the delay between hypostome determination and tentacle regeneration was not as great as in the case of FU-treated animals. After a 20 h pre-treatment, HU-treated hydra developed tentacle buds within 24 h of cutting at the subhypostomal level. This delay of approximately 19 h between hypostome formation and tentacle morphogenesis is similar to that found in untreated controls (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 the subhypostomal level, placed in fresh actinomycin medium, and at various times thereafter the distal tip of a reconstituting animal was transplanted to the digestive zone of an untreated host hydra. Results are shown in Table 8. Actinomycin D treatment caused quite a considerable delay in the time required for the grafts to become determined: T50 = 10 h. The term 'hypostome determination' has not been applied to these results because, in striking contrast to the behaviour of control subhypostomal grafts, the great majority of secondary axes induced after actinomycin treatment were in the form of a typical peduncle and basal disk. Thus twenty-two of the twenty-four secondary axes induced after actinomycin treatment were of this type, and only one type 1 Pattern regulation in hydra. II 63 induction was obtained as a result of grafting at 6 h after cutting and another type 1 induction at 16 h. After a 4 h pre-treatment, animals continuously treated with actinomycin D at 10 /*g/ml disintegrated within 24-36 h of cutting at the subhypostomal level without showing any signs of tentacle regeneration. In view of the type of secondary axes induced by grafts from actinomycin Dtreated animals, it is also worth noting that these animals showed no signs of the formation of proximal regions at the distal end. Table 8. Effect of actinomycin D on hypos tome formation from the subhypostomal region Source of graft Control subhypostome Actinomycin D-treated subhypostome Time after cutting when grafted (h) No. of successful grafts No. of animals with secondary axis 6 19 14 21 22 14(74%) 0 7(33%) 17(77%) 0-4 6 16 Table 9. Effect of chloramphenicol onhypostome formation from the subhypostomal region Source of graft Control subhypostome Chloramphenicol-treated subhypostome Time after cutting when grafted (h) No. of successful grafts No. of animals with secondary axis 6 0-i 6 16 24 15 12 20 18 19 12(80%) 0 0 1 (6%) 4 (21 %) (d) Effect of chloramphenicol on hypostome formation The procedure in this experiment was the same as that in the last except actinomycin D was replaced by chloramphenicol at 600 /*g/ml and that a 20 h pre-treatment was employed. The number of animals producing secondary axes after transplantation of control and chloramphenicol-treated subhypostomes is shown in Table 9. No T50 was obtained for the chloramphenicol-treated subhypostomes because even after 24 h only a small number of transplanted pieces induced. The secondary axes that were obtained were all type 1 or 3 inductions. The experiment was not continued beyond 24 h for the animals died within 24-36 h of cutting at the subhypostomal level during chloramphenicol treatment, disintegration invariably commencing at the distal end. This experiment shows therefore that chloramphenicol treatment causes a very considerable delay in the time 64 S. G. CLARKSON required for hypostome determination from the subhypostomal region. While no T50 could be obtained for chloramphenicol-treated subhypostomes, it is evident that this delay is at least 20 h. DISCUSSION Some consideration must be given to the inhibition of nucleic acid and protein synthesis by the four compounds during the time required for the grafts to become determined in the transplantation experiments. After a 4 or 20 h pretreatment period, continuous treatment of regenerating animals with the compounds at the above concentrations results in their disintegration within 24-48 h of cutting at the subhypostomal level. It is considered extremely unlikely, therefore, that nucleic acid and protein synthesis would recover beyond the first hour of distal regeneration under these conditions, and it is assumed that during the time required for the grafts to become determined in the transplantation experiments the extent of inhibition of DNA, RNA, or protein synthesis in the treated samples is at least the same as that recorded within the first hour of cutting at the subhypostomal level. This has been shown to be the case 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 transplantation to untreated host hydra. Since the pieces were transplanted to the same region of the host, the formation of a secondary axis as a result of transplantation depends upon changes in the transplanted piece, specifically the acquisition of inductive ability and resistance to absorption (Webster & Wolpert, 1966). The nature of the test implies, however, that these properties are acquired before transplantation is performed, i.e. while the prospective grafts are still being treated with one of the inhibitors. It is suggested therefore that the possible recovery of DNA, RNA or protein synthesis in the grafted pieces cannot account for the formation of secondary axes in the transplantation experiments. The finding that HU severely inhibits the incorporation of [3H]thymidine into DNA in regenerating hydra while having little effect on [3H]uridine incorporation into RNA is in accord with the work on the effects of this compound on HeLa cells (Young & Hodas, 1964), ascites tumour cells (Yarbro et al. 1965), and regenerating rat liver (Schwartz et al. 1965). Under conditions of HU treatment that cause a 60 % reduction in [3H]thymidine incorporation (Table 3), the time required for hypostome determination from the subhypostomal region is increased by only 22 % (4£to 5\ h, Table 7). It is therefore concluded that DNA synthesis plays no major role in the acquisition of organizing properties by the hypostome. At first sight the data on the effect of FU on distal regeneration appear to refute this conclusion. Thus, FU causes a threefold increase in the time required for hypostome determination (4^ to 13^ h, Table 6) and, on the basis of the Pattern regulation in hydra. II 3 3 65 inhibition of [ H]thymidine and [ H]cytosine incorporation (Tables 1, 2), it is assumed 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 time for hypostome formation is unlikely to be due solely to their different levels of inhibition of DNA synthesis. Rather, it seems more reasonable to suggest that the T50 of 13£h for FU-treated subhypostomes, when compared to the 5^h T50 of HU-treated subhypostomes, reflects the fact that [3H]uridine incorporation 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 protein synthesis, for FU slightly inhibits protein synthesis in addition to, or as a consequence of, its effect on RNA synthesis (Table 1). It is therefore concluded that the delay of hypostome formation in FU-treated hydra is not a consequence of the inhibition of DNA synthesis, but rather of the inhibition of RNA and possibly also protein synthesis. This is consistent with the earlier suggestion (Clarkson, 1969) that RNA and protein synthesis play important roles in the determination of a hypostome. Actinomycin D inhibits RNA synthesis by 85 % during the first hour of distal regeneration following a 4 h pre-treatment (Table 4) and it is assumed that this level of inhibition is at least maintained during the time required for the actinomycin D-treated grafts to become determined in the transplantation experiments shown in Table 8. The customary action of actinomycin is to inhibit DNA-primed RNA synthesis, and the nature of the residual 15% actinomycin-resistant fraction is therefore important for the interpretation of the effects of this compound at a biochemical level. If this 15 % incorporation reflects even a small amount of synthesis of an active messenger RNA fraction, it could account for a great deal of protein synthesis and perhaps have important developmental effects. On the other hand, there are at least two other possible explanations for this 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 RNA after conversion of uracil to cytosine (Franklin, 1963). While it remains to be demonstrated that actinomycin D inhibits the synthesis of RNA molecules large enough to be thought of as normal messenger RNA in hydra, the finding that the incorporation of [14C]algal protein hydrolysate into protein is inhibited by only 23 % after RNA synthesis has been substantially inhibited (Table 4) strongly suggests that relatively stable messenger RNAs exist in hydra. At a biological level the effect of actinomycin D is extremely interesting. The transplantation experiments indicate that hypostome formation is severely inhibited by actinomycin, at least as judged by the small number of type 1 induced secondary axes. On the other hand, resistance to absorption was acquired within a T 50 of 10 h, the great majority of the induced axes being in the form 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 is interesting to note that a dissociation of these two properties has been suggested by Webster (1967) from a quite different approach. If the resistance to absorption normally displayed by the determined hypostome is dependent on resistance to inhibition and therefore upon high threshold (Webster, 1967), the fact that resistance to absorption can be acquired in the presence of actinomycin D suggests that either the 15% actinomycin-resistant RNA fraction is actually all the template material necessary for the acquisition of high threshold, the inhibited fraction being somehow irrelevant, or threshold is predominantly under translation control. It is also possible, although less likely, that this factor is independent of all RNA synthesis. Conversely, the inability of actinomycin D-treated subhypostomes to induce type 1 secondary axes following transplantation might suggest that the normal acquisition of inductive ability by the hypostome involves dramatic changes in transcription. An alternative explanation for the secondary axes induced by actinomycin D-treated subhypostomes is possible if it is assumed that they reflect the determination of a basal disk at the distal end. It is known that a piece of basal disk retains its identity when transplanted (Burt, 1925; Burnett, 1959), although it has been suggested that the induced basal disks are made entirely from the graft tissue (Burnett, 1961), unlike the induction of a secondary axis by a hypostome which utilizes host tissue (Yao, 1945). Whether or not host material is incorporated into the induced basal disk, it seems clear that this region possesses the ability to resist absorption. In addition, a basal disk can form at the distal end of regenerating hydra under some circumstances, e.g. treatment with Cleland's reagent (S. G. Clarkson, unpublished observations). Whether a similar situation exists in the case of actinomycin treatment is difficult to determine for animals kept in actinomycin disintegrated within 24-36 h of cutting at the subhypostomal level without showing signs of either distal or proximal structures at the distal end. It should be noted, however, that basal disk formation from the proximal end of hydra reconstituting in ' M ' requires about 20 h (Webster, 1964). It is therefore difficult to envisage how a basal disk could form within such a short time as 10 h at the distal end of the axis. Moreover, the secondary axes obtained in the present study were significantly larger than the size of the implanted pieces. This suggests that some reorientation of host material did occur and, therefore, that the induced axes reflect the resistance to absorption of the hypostome rather than the determination of a basal disk at the distal end. It will be evident that certain difficulties arise when the effects of FU and actinomycin D on RNA synthesis and hypostome determination are compared. It was suggested earlier that FU causes a threefold increase in the time required for hypostome determination by virtue of its inhibition of RNA rather than DNA 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 67 absorption whereas FU-treated subhypostomes acquire both resistance to absorption and inductive ability. The results are therefore difficult to correlate when put in terms of total RNA synthesis. In addition, there exists no completely satisfactory explanation of the inhibition of RNA synthesis by FU, although Heidelberger (1965) has made the interesting proposal that FU incorporated into messenger RNA does not affect the process of translation whereas FU does appear to have an inhibitory effect on transcription, possibly through its appearance at sites on messenger RNA normally occupied by cytosine. The finding 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 as meaning that a large proportion of protein synthesis during early distal regeneration is coded for by messengers of relatively long half-lives. In spite of ignorance of the detailed biochemical effects of FU it would thus appear that the delay of distal regeneration in FU-treated subhypostomes is due primarily to inhibition of RNA synthesis. On the other hand, chloramphenicol severely inhibits [14C]algal protein hydrolysate incorporation into protein (Table 5) and under these conditions hypostome determination is delayed by at least 20 h (Table 9). This suggests that protein synthesis plays a major role in the acquisition of resistance to absorption and inductive ability by the hypostome, although the results may be due more simply to the toxicity of chloramphenicol to hydra. While inhibition of bacterial protein synthesis by this compound has been amply documented, conflicting observations have 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 explanation is possible of the delay of hypostome determination by chloramphenicol. In spite of the inherent limitations of this level of analysis, the results are consistent with the earlier suggestion (Clarkson, 1969) that RNA and protein synthesis play important roles in the control of polarized regulation in hydra, and with the recent evidence which suggests that growth plays no major role in this process (Campbell, 1967; Clarkson & Wolpert, 1967; Webster, 1967; Clarkson, 1969). The experiments with actinomycin D in particular suggest that an important proportion of protein synthesis in hydra is performed utilizing relatively stable RNA templates. Thus, threshold might be predominantly under translational control and this perhaps could account for the assumed stability of this factor (Webster, 1966a, b). This is also consistent with the view that the stimulus for reconstitution is not 'activation', but release from inhibition (Rose & Rose, 1941; Webster, 1966«). Conversely, the synthesis of RNA molecules having only a limited half-life could provide the means of control for a transitory response such as tentacle morphogenesis. These suggestions must be regarded as extremely tentative, however, for it has yet to be demonstrated that protein synthesis in hydra is of the sort that requires template RNA, i.e. polysomal, or indeed that the sedimentation profiles of hydra RNA are similar to those of other animal cells. 5-2 68 S. G. CLARKSON SUMMARY 1. The effects of inhibition of nucleic acid and protein synthesis on the time required for hypostome formation from the subhypostomal region were investigated using biochemical and transplantation techniques. 2. Hydroxyurea can cause a 22 % increase in the time required for hypostome formation. The incorporation of [3H]thymidine into DNA is severely inhibited under these conditions, whereas RNA synthesis appears to be essentially normal. 3. 5-fluorouracil severely inhibits the incorporation of both [3H]thymidine and [3H]cytosine into DNA, and the incorporation of [3H]uridine into RNA under conditions that apparently produce only a slight inhibition of protein synthesis. The time for hypostome determination is increased threefold under these conditions. This delay is considered to be due primarily to the inhibition of RNA synthesis. 4. Actinomycin D substantially suppresses [3H]uridine incorporation into RNA but has only a slight inhibitory effect on [14C]algal protein hydrolysate incorporation into protein. Hypostome formation is not completely inhibited under these conditions for actinomycin D-treated subhypostomes can acquire resistance to absorption. The majority of the hypostomes formed, however, do not possess normal inductive ability. This suggests that the factors responsible for the inductive ability of the hypostome (i.e. tentacle morphogenesis) are distinct from those controlling resistance to absorption. This might reflect differences in the stability of messenger RNAs. 5. Chloramphenicol severely inhibits [14C]algal protein hydrolysate incorporation into protein. The time for hypostome determination appears to be delayed by at least 20 h under these conditions, but this may reflect the toxicity of chloramphenicol to hydra. 6. The significance of the results is discussed in relation to concepts developed by other workers to explain polarized regulation in hydra. RESUME La synthese des acides nucleiques et des proteines et la regulation de la morphogenese chez Vhydre. II. Les ejfets de Vinhibition de la synthese des acides nucleiques et des proteines sur la formation de Vhypostome 1. Des techniques biochimiques et des transplantations ont ete utilisees pour etudier les effets de l'inhibition de la synthese des acides nucleiques et des proteines sur la duree de formation de la region orale (hypostome) de l'hydre, a partir de la region sub-orale. 2. La duree de formation de l'hypostome est augmentee de 22% par un traitement a l'hydroxyuree: l'incorporation de [3H]thymidine dans le DNA est nettement inhibee dans ces conditions, alors que la synthese du RNA reste relativement normale. Pattern regulation in hydra. II 3 69 3 3. L'incorporation de H-thymidine et de H-cytosine dans le DNA, de meme que celle de la 3H-uridine dans le RNA, est tres fortement reduite en presence de 5-fluorouracil, alors que la reduction de la synthese proteique n'est que partielle. Le temps de formation de l'hypostome est triple dans ces conditions. 4. L'actinomycine reduit tres fortement l'incorporation de 3H-uridine dans le RNA, mais n'a qu'un tres leger effet inhibiteur sur l'incorporation d'un hydrolysat de proteines-C14. L'inhibition de la formation de l'hypostome n'est cependant pas complete, puisqu'apres un traitement a l'actinomycine, une resistance a l'absorption de cet antibiotique se developpe. La plupart des hypostomes formes dans de telles conditions experimentales ne possedent cependant pas une capacite inductrice normale. Les facteurs responsables du pouvoir inducteur et controlant la morphogenese des tentacules seraient done de nature differente de ceux influencant la resistance a l'absorption. II est possible que ce soit le reflet de differences de stabilite au niveau des mRNAs. 5. Le chloramphenicol inhibe nettement l'incorporation d'hydrolysat de proteines-C14 dans les proteines. Un retard de 20 h s'observe dans le developpement de l'hypostome en presence de cet antibiotique, mais il n'est pas exclu que cet effet soit du a la toxicite du produit. 6. La signification de ces resultats est discutee par rapport aux differentes hypotheses deja emises par d'autres auteurs pour expliquer la regulation de la morphogenese chez l'hydre. I am deeply indebted to Professor Lewis Wolpert for his advice and encouragement. I am grateful to Squibbs and Sons Ltd. for a gift of hydroxyurea, and Dr S. J. Coward for a gift of actinomycin D. We wish to thank the Agricultural Research Council for a scintillation counter, and the Nuffield Foundation for their support of this work. BURNETT, BURNETT, BURNETT, REFERENCES A. L. (1959). Histophysiology of growth in hydra. /. exp. Zool. 140, 281-342. A. L. (1961). The growth process in hydra. /. exp. Zool. 146, 21-84. A. L. (1966). A model of growth and cell differentiation in hydra. Am. Nat. 100, 165-90. D. R. R. (1925). 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