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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.
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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,
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(Manuscript received 30 April 1968)