/. Embryol. exp. Morph. Vol. 21, J, pp. 33-54, February 1969
Printed in Great Britain
33
Nucleic acid and protein synthesis and pattern
regulation in hydra
I. Regional patterns of synthesis and changes in synthesis
during hypostome formation
By S. G. CLARKSON 1
From the Department of Biology as Applied to Medicine,
Middlesex Hospital Medical School, London
Hydra provides a convenient system for the study of the regulation of a
linear pattern of organization. Almost any region is capable of being reconstituted into the whole organism and regulation is always polarized, i.e. distal
structures (hypostome and tentacles) are formed from distal ends and proximal
structures (peduncle and basal disk) from proximal ends.
Two models have recently been proposed to account for polarized regulation
in hydra. Both incorporate axial gradients, but they differ radically from each
other and some controversy exists as to which formulation is correct. In the
model of Webster (Webster, 1966tf, b; Webster & Wolpert, 1966) regulation
occurs as the result of the interaction of three factors, namely time for hypostome determination, inhibition of hypostome determination, and threshold for
inhibition. No suggestions are made with regard to the physiological basis of
these factors. On the other hand, Burnett (1961, 1966) claims a close causal
relationship between growth and form. In his model, regulation is determined
by the local balance of two factors, a growth stimulator arising from the
hypostome, and a growth inhibitor produced by regions of active growth.
One approach towards an understanding of the physiological basis of the
proposed axial gradients and of the factors involved in regulation is the
determination of nucleic acid and protein synthesis in intact and regenerating
hydra. While the axial distributions of DNA, RNA, and protein have been
studied in a few hydroid species, e.g. Hydra littoralis (Li & Lenhoff, 1961) and
Tubularia larynx (Tardent, 1963), no reports exist of the quantitative measurement of their synthesis. This provides the subject of this paper. Essentially two
questions were asked: (1) Does hydra possess axial gradients of DNA, RNA, or
1
Author's address: Department of Molecular, Cellular and Developmental Biology,
University of Colorado, Boulder, Colorado 80302, U.S.A.
3
JEEM 21
34
S. G. CLARKSON
protein synthesis? (2) Is the formation of a hypostome during regulation
accompanied by radical changes in DNA, RNA, or protein synthesis?
MATERIALS AND METHODS
Hydra Httoralis were used for all experiments. Details with regard to culture
methods, selection of animals, and operative procedures are as given in Webster
& Wolpert (1966).
Radioactivity labelling procedures. Radioactive precursors were administered
by incubating intact or regenerating hydra with precursor in culture medium
' M ' (Muscatine, 1961). In some experiments precursor was administered in the
presence of 10~ 5 M reduced glutathione (GSH) in ' M ' , because GSH at this
concentration causes the hypostome to open wide for 35 min (Loomis, 1955),
thereby giving endodermal cells better access to the precursor. The hydra were
incubated at 26 °C as batches of five or six in 0-2 ml or ten in 0-5 ml of labelled
medium.
[3H]thymidine (3 or 5 c/mM), [3H]uridine (3-33 c/mM), and [14C]algal protein
hydrolysate (640^c/mg) were obtained from the Radiochemical Centre,
Amersham.
Radioactivity determination. Incubations were terminated by washing the
hydra four times with an excess of unlabelled precursor in ' M ' solution prechilled to 4 °C. Debris was removed from the basal disks and, in regional
incorporation studies, the animals were cut in the unlabelled precursor medium
and identical regions pooled. The samples were disrupted by sonication under
ice (Dawe Soniprobe, setting no. 3,5 s burst), aliquots removed for DNA, RNA,
or protein estimation, and cold 10 % trichloroacetic acid added to the remaining
homogenate. In studies utilizing [14C]algal protein hydrolysate the samples were
heated at 90 °C for 20 min to hydrolyse nucleic acids, and then cooled by the
addition of cold 5 % trichloroacetic acid. Acid-insoluble radioactive material
was assayed by collecting the precipitate on a Millipore filter (type GS, 25 mm,
pore diameter 0-22 /*), washing thoroughly with cold 5 % trichloroacetic acid,
briefly rinsing with a 2:1 mixture of chloroform:isopropanol and then ether,
and counting in a Packard TriCarb scintillation counter in a toluene-based
scintillant containing PPO (2,5-diphenyloxazole), 5 g/1., and dimethyl-POPOP
(4-bis-[4-methyl-5-phenyloxazoyl-2]-benzene), 0-3 g/1. Corrections were made
for 3 H self-absorption, but 14C self-absorption was negligible. All radioactivity
is reported as counts/min per jug nucleic acid or protein. This is based on the
assumption that each radioactive precursor was incorporated into the relevant
portion of the acid-insoluble fraction.
Protein and nucleic acid estimation. Protein determinations were made
directly on aliquots of the homogenate by the method of Lowry, Rosebrough,
Farr & Randall (1951). Wellcome Chemical Control Serum (Burroughs Wellcome and Co., London) was used as standard.
Pattern regulation in hydra. I
35
Nucleic acids were extracted by the addition of an equal volume of cold 10%
trichloroacetic acid to aliquots of the homogenate, followed by centrifugatjon at
4 °C. DNA was extracted from the precipitate either by a single treatment with
1 N-NaOH for 1 h at 37 °C, or by two 20 min extractions with 1-8N perchloric
acid at 70 °C. DNA determinations were made directly on the alkali digest, or on
the combined acid extracts, by the indole-HCl method of Ceriotti (1955) as
modified by Bonting & Jones (1957). Calf thymus DNA (Sigma Chemical Co.
London) was used as standard.
RNA was extracted from the precipitate by hydrolysis in 0-3N-KOH for 1 h at
37 °C. After cooling under ice, each sample was acidified with an equal volume
of cold 0-4N perchloric acid and allowed to stand for 10 min at 0 °C. Following
centrifugation, the precipitate was washed with 1-vol. of cold 0-2N perchloric
acid and centrifuged again. The two supernatants were combined and estimated
for RNA by u.v. absorption at 260 m/.t. Yeast RNA (Sigma Chemical Co.,
London) was used as standard.
RESULTS
1. Conditions of radioactive precursor incorporation
It has generally been thought that hydra are impermeable to most exogenous
free compounds. Burnett, Baird & Diehl (1962) were forced to cut hydra in
order to obtain incorporation of [3H]thymidine, and Campbell (1965) devised a
microinjection procedure for the same purpose. However, initial experiments
involving incubation of intact hydra with [3H]thymidine administered as a
component of the normal culture medium indicated that this precursor could be
incorporated into the acid-insoluble fraction to quite a large extent, the great
majority of the radioactive material presumably being incorporated into DNA.
In view of this discrepancy the incorporation of [3H]thymidine, [3H]uridine, and
[l4C]algal protein hydrolysate was investigated.
(a) Incorporation with time
To establish the kinetics of [3H]thymidine incorporation, triplicate batches of
six intact hydra were incubated in 50/*c/ml [3H]thymidine (5 c/raivi) in ' M ' for
the times indicated (Fig. 1A). Similar experiments were performed with
[3H]uridine (3-33 c/raM) at 15 /*c/ml in ' M ' (Fig. 1B) and with [14C]algal protein
hydrolysate (640/^c/mg) at 10//c/ml in ' M ' (Fig. 1C). Regression lines have
been calculated for the results shown in Fig. 1; in each case the relationship is
linear (correlation coefficient, r < 0-001) and the intercepts on the x and y axes
are not significantly different from zero. Non-specific absorption of any of the
three precursors is considered negligible, for animals incubated for 5 min
possessed specific activities which were no higher than the values predicted from
the equation of their respective regression line for a 5 min incubation. It is
therefore concluded that hydra can incorporate [3H]thymidine, [3H]uridine, and
3-2
36
S. G. CLARKSON
[ C]algal protein hydrolysate from the surrounding medium at a constant rate
for at least the times investigated.
14
(b) Incorporation at different precursor concentrations
To determine how the rate of incorporation of [3H]thymtdine varies as a
function of the concentration of externally supplied [3H]thymidine of constant
specific activity, duplicate batches of five intact hydra were incubated for 2\ h in
3
2
6 9
18
Incubation time (h)
120
I
-c
_
2
4
6
Incubation time (h)
8
/
-
'p
/
o.
Counts/i
24
I
/
| 90
o
^60
1
4
6
8
Incubation time (h)
/
i
i
i
Fig. 1. Kinetics of (A) [3H]thymidine incorporation into DNA, (B) [3H]uridine
incorporation into RNA, and (C) [14C]algal protein hydrolysate incorporation into
protein.
[3H]thymidine (5 c/mM) in 10~ 5 M GSH in ' M ' at various concentrations between
3-125 and 50/tc/ml. Similar experiments were performed with [3H]uridine
(3-33 c/mM) in ' M ' over the range 3-125-50/^c/ml, and with [14C]algal protein
hydrolysate (640 ^c/mg) in' M ' over the range 3-20 /ic/mi. The results are shown
in Fig. 2, together with the regression lines (r < 0-001 in each case). The inter-
Pattern regulation in hydra. I
37
cepts on the x and y axes are not significantly different from zero, and it is
evident that the rate of incorporation of each precursor is a linear function of
the external precursor concentration over the range investigated.
140
7 120 -
7-5 15
30
Concentration of
[ H]thymidine (/<c/m
60
c1
1
6-25125
25
Concentration
of
3
[ H]uridine (/ic/m\)
3
1
•
o
M60
I45
-
-
-
-
/
/
c
3 30 -
-
15
•''
1
1
1
1
3 6
12
20
Concentration of [14C]algal
protein hydrolysate (//c/ml)
Fig. 2. Influence of (A) [3H]thymidine concentration on its incorporation into DNA,
(B) [3H]uridine concentration on its incorporation into RNA, and (C) [14C]algal
protein hydrolysate concentration on its incorporation into protein.
2. Reduced glutathione as a specific means to increase radioactive precursor
incorporation
Preliminary experiments involving incubation of intact hydra with [3H]thymidine in 10~ 5 M GSH in ' M ' indicated that incorporation could be increased
some threefold over that obtained by incubating hydra with [3H]thymidine in
' M ' alone. The following experiments were designed to test if this increased
incorporation was due specifically to the glutathione feeding reflex.
38
S. G. CLARKSON
(a) DNA synthesis
In the first experiment, four groups of duplicate batches of five animals were
incubated for 2^h in [3H]thymidine (5 C/IUM) at 50/tc/ml. Groups (1) and (2)
comprised intact animals; groups (3) and (4) comprised animals cut at the
subhypostomal level immediately prior to incubation: both distal and proximal
parts were retained for incubation. Groups (1) and (3) were incubated in
[3H]thymidine in ' M ' alone; groups (2) and (4) were incubated in [3H]thymidine
in 10~ 5 M GSH in ' M \ The results are shown in Table 1.
Table 1. Reduced glutathione as a specific means to increase
the incorporation of [3H]thymidine into DNA
Treatment and materials
(1) Intact hydra
[3H]TDR alone
(2) Intact hydra
[3H]TDR+10-5MGSH
(3) Subhypo. cut hydra
[3H]TDR alone
(4) Subhypo. cut hydra
[3H]TDR + 1 0 - 5 M G S H
Specific activity
(c.p.m.//*g DNA)
Mean specific
activity
277,
296/
1122j
916/
886 \
805/
968 \
1015/
286
1019
845
992
A significant threefold increase was obtained by incubating intact hydra in
the presence of GSH compared to that obtained by incubation in [3H]thymidine
in ' M ' alone: applying Student's t test, P < 0-05 for the difference between the
means of groups (1) and (2). Reduced glutathione presumably could cause this
increase by allowing the cells of the endoderm better access to the thymidine by
means of the feeding reflex and/or by establishing a complex biochemical
situation which somehow allowed more incorporation of the precursor. By
removing the hypostome and tentacles just before incubation, however, the
feeding reflex would be prevented while still allowing endodermal cells better
access to the precursor. If the latter were the major factor responsible for the
enhanced incorporation using GSH, then animals cut at the subhypostomal level
should show no significant difference between their specific activities when
incubated in the presence or absence of GSH. This was in fact found to be the
case: P > 0-10 for the difference between the means of groups (3) and (4). It is
therefore concluded that the increased incorporation obtained when hydra are
incubated in the presence of GSH is not due to a non-specific biochemical effect,
but rather that it reflects better access given to the cells of the endoderm to the
[3H]thymidine by means of the GSH feeding reflex.
In addition, it is evident from Table 1 that cutting at the subhypostomal level
leads to a very significant increase in incorporation: P < 0-02 for the difference
Pattern regulation in hydra. I
39
between the means of groups (1) and (3). Presumably this could be due to a
stimulation of DNA synthesis on cutting and/or the fact that endodermal cells
are again given better access to the precursor. Incubation of intact animals in the
presence of GSH, group (2), produces similar specific activities to those of
animals cut at the subhypostomal level incubated in [3H]thymidine in ' M ' alone,
group (3). However, an increase in the availability of precursor to endodermal
cells is considered the major factor for the increased incorporation with GSH
and, on this basis, it would therefore seem unlikely that cutting stimulates DNA
synthesis. This conclusion is further borne out by a consideration of groups (2)
and (4), for animals cut at the subhypostomal level incubated in the presence of
GSH do not show a significant increase in incorporation when compared to
intact animals similarly incubated: P > 0-80 for the difference between the
means of groups (2) and (4).
The most obvious difficulty in evaluating the above results is that one of the
likely consequences of the feeding reflex is mechanical stretching of cells of the
hypostome. This, in turn, could lead to localized permeability changes and
could possibly explain the slightly higher incorporation obtained by intact
animals incubated in GSH, group (2), compared to that obtained by incubating
animals cut at the subhypostomal level in GSH, group (4). If this were the case,
then changes in permeability of the cells of the hypostome would be relatively
unimportant. A more serious alternative, however, is the possibility that cutting
does stimulate DNA synthesis and that this stimulation is balanced by that due
to permeability changes. The present experiment does not allow us to distinguish
between these alternatives, and the conclusion that cutting does not stimulate
DNA synthesis should therefore be accepted, for the moment, with some
caution.
(b) RNA synthesis
The procedure in this experiment was exactly the same as that in the previous
one except that [3H]uridine (3-33 c/mM) at 10/ic/ml was substituted for
[3H]thymidine. The specific activities of the four groups are shown in Table 2.
Using the same rationale as was applied to the previous experiment, the
significantly increased incorporation obtained by incubating intact animals in
the presence of GSH (P < 0-01 for the difference between the means of groups
(1) and (2)) is considered to be due to the GSH feeding reflex, for there is no
significant difference between the incorporation obtained for animals cut at the
subhypostomal level incubated in the presence or absence of GSH {p > 0-70 for
the difference between the means of groups (3) and (4)).
Table 2 also demonstrates that animals cut at the subhypostomal level show a
marked increase in [3H]uridine incorporation (P < 0-01 for the difference
between the means of groups (1) and (3)) which may reflect a stimulation of
RNA synthesis on cutting and/or an increased availability of [3H]uridine to
endodermal cells. Since animals cut at the subhypostomal level incubated in
40
S. G. CLARKSON
3
[ H]uridine in ' M ' alone, group (3), possess much higher specific activities than
do intact animals incubated in the presence of GSH, group (2), and since better
access of endodermal cells to the precursor is a feature common to both groups,
it may be suggested that cutting does stimulate RNA synthesis. However, there
remains the possibility that cutting leads to an even greater availability of the
precursor than is attained by the GSH feeding reflex through, for example, the
[3H]uridine entering the cells at the cut surface and traversing the mesoglea.
This could account perhaps for the difference between groups (2) and (4):
animals cut at the subhypostomal level incubated in the presence of GSH
Table 2. Reduced glutathione as a specific means to increase
the incorporation of[3H]uridine into RNA
Treatment and materials
(1) Intact hydra
[3H]UDR alone
(2) Intact hydra
[3H]UDR+10-5MGSH
(3) Subhypo. cut hydra
[3H]UDR alone
(4) Subhypo. cut hydra
[3H]UDR+10-5MGSH
Specific activity
(c.p.m.//*g RNA)
23-1 \
25-5/
62-3i
58-5/
212-6 \
239-7 /
253-6 \
222-0 /
Mean specific
activity
24-3
60-4
226-2
237-8
possess a significant (P < 0-02) increase in incorporation compared to intact
animals similarly incubated. However, if an incorporation pathway via the cut
surface were a major factor, it is surprising that there was no significant difference between groups (2) and (4) of the comparable experiment with [3H]thymidine (Table 1), for uridine and thymidine have similar molecular weights.
Moreover, the difference between groups (2) and (4) in the present experiment
represents nearly a 300% increase in [3H]uridine incorporation. In view of the
magnitude of this increase, it is considered likely that cutting does stimulate
. RNA synthesis.
(c) Protein synthesis
An experiment identical to the previous one was performed except that the
incorporation of [14C]algal protein hydrolysate (640/^c/mg) at 10/*c/ml into
protein of the four groups was measured. Results are shown in Table 3.
The results demonstrate a significant difference (P < 0-01) between the means
of groups (1) and (2), but not (P > 0-30) between the means of groups (3) and
(4). It is therefore concluded that the enhanced incorporation obtained as a
result of incubating intact animals in the presence of GSH is due primarily to the
fact that cells of the endoderm are given better access to the precursor by means
of the GSH feeding reflex.
Pattern regulation in hydra. I
41
This experiment further demonstrates a significant difference (P < 0-02)
between the means of groups (1) and (3), and a slightly significant difference
(P < 0-10) between the means of groups (2) and (4). The latter difference
represents only a 25 % increase in the incorporation of [14C]algal protein
hydrolysate and, while this might indicate that cutting does stimulate protein
synthesis, the possibility cannot be excluded that part or all of this increased
incorporation is due to the easier entry of the precursor. It would be surprising,
however, if the large increase in RNA synthesis obtained under identical
conditions (Table 2) were not accompanied by some increase in protein synthesis,
and for this reason it is felt that the apparent stimulation of protein synthesis
following cutting is probably genuine.
Table 3. Reduced glutathione as a specific means to increase the
incorporation of [uC]algal protein hydrolysate into protein
Treatment and materials
(1) Intact hydra
[14C]hydrolysate alone
(2) Intact hydra
[14C]hydrolysate+10-5 M GSH
(3) Subhypo. cut hydra
[14C]hydrolysate alone
(4) Subhypo. cut hydra
[14C]hydroylsate+10~5 M GSH
Specific activity
(c.p.m.//*g protein)
36-7 \
33-6/
92-7»
860/
96-2 \
105-4/
107-81
114-7/
Mean specific
activity
35-2
89-4
100-8
111-3
3. Regional patterns of nucleic acid and protein synthesis
(a) DMA synthesis
In the first experiment, ten intact hydra were incubated for 24 h in [3H]thymidine (3 c/mM) at 12-5 /*c/ml in ' M ' , cut into regions which were pooled and
their specific activities determined. Results of three experiments are shown in
Table 4. Since the information required is the pattern of incorporation rather
than the absolute activities, the mean specific activity of each region has been
determined from the three experiments and each one is expressed as a percentage
of the maximum mean specific activity of the five regions.
The pattern which emerges from these experiments is a broad distribution of
activity with a very slight peak in the bud. The hypostome and tentacles and the
peduncle and basal disk possess a somewhat lower activity, but the activity of the
gastric region is approximately constant.
One difficulty in evaluating these results is that the digestive zone is of far
greater size and contains many more cells than any of the other four regions. It is
possible therefore that the over-all activity of the digestive zone measured in
these experiments could be masking a significant difference between a 'sub-
42
S. G. CLARKSON
hypostomal growth zone' and the remainder of the digestive zone. The following
experiment was designed to investigate this possibility.
Twelve intact hydra were incubated in [3H]thymidine under conditions
identical to the previous experiment. At the end of 24 h the digestive zones were
isolated and cut into thirds, identical regions pooled, and their specific activities
determined. Results of three experiments are shown in Table 5.
Table 4. Regional incorporation of [3H]thymidine
into DNA of budding hydra
Specific activity (c.p.m.//<tg DNA)
Exp. 1
Exp. 2
Exp. 3
Mean
specific
activity
1651
1941
196-5
202-7
260-5
280-5
260-5
281-9
2280
230-6
231-3
254-2
217-9
2351
229-4
246-3
88
95
93
100
133-5
226-4
185-6
181-8
74
A
Regions
Hypostome and
tentacles
Digestive zone
Budding zone
Bud
Peduncle and
basal disk
%of
maximum
mean
specific
activity
Table 5. Incorporation of[3H]thymidine into DNA
within the digestive zone
Exp. 3
Mean
specific
activity
%of
maximum
mean
specific
activity
3951
420-9
326-3
450-9
429-5
447-1
100
95
99
Specific activity (c.p .m.//*g DNA)
Regions of the
digestive zone
Exp. 1
Exp. 2
Distal third
Middle third
Proximal third
484-1
473-4
521-6
473-5
3941
493-4
It is evident that there are no major variations in the pattern of thymidine
incorporation within the digestive zone. Thus, unless there is a highly localized
'subhypostomal growth zone', the over-all activity of the digestive zone
measured in the previous experiments does reflect an approximately uniform
incorporation of [3H]thymidine along its length.
One criticism that can be levelled against both experiments, however, is that
an incubation time of 24 h may be too long, and that this length of time allows
the [3H]thymidine to be 'soaked up' by cells of all regions, thus resulting in the
apparently broad distribution of activity. One way of circumventing this
problem is to use a shorter incubation period in conjunction with another means
Pattern regulation in hydra. I
43
of administering the precursor. The following experiment was therefore
performed.
Twenty animals were incubated for \ h in [3H]thymidine (3 c/misi) at 50 /tc/ml
in 10~5M GSH in ' M ' , cut into regions which were pooled and their specific
activities determined. Results are shown in Table 6.
Table 6. Regional incorporation of [3H]thymidine into DNA of
hydra incubated in the presence of reduced glutathione
Regions
Hypostome and
tentacles
Digestive zone
Budding zone*
Peduncle and
basal disk
Specific activity
(c.p.m.//*g DNA)
%of
maximum
specific activity
61-97
59-98
5510
100
97
89
38-44
62
* Twenty buds were also isolated but were lost during centrifugation.
The results from this pulse-labelling experiment indicate an approximately
constant activity within the gastric region and a somewhat lower activity in the
peduncle and basal disk. Comparison with the data from a 24 h incubation
period without GSH shows that the only change in the pattern of specific
activities is that the hypostome and tentacles possess greater activity than the
digestive zone during a brief label with GSH (Tables 4, 6). The simplest way of
explaining this slightly altered pattern is to postulate that the increased activity
at the distal end reflects some localized permeability changes associated with the
feeding reflex. This, of course, would indicate that such changes are not major
contributory factors to the increased incorporation obtained by incubating
intact animals in the presence of GSH.
These three experiments indicate therefore that DNA synthesis is almost
uniformly distributed throughout the body column.
(b) RNA synthesis
Ten intact hydra were incubated for 2 h in [3H]uridine (3-33 c/mivi) at
25 /fc/ml in ' M ' , cut into regions which were pooled and their specific activities
determined. The results of three experiments are shown in Table 7.
The pattern emerging from these experiments is an axial gradient of incorporation of [3H]uridine. In all three experiments the maximum specific activity is
found in the hypostome and tentacles, the second highest activity in the digestive
zone, and the lowest in the peduncle and basal disk. The relative activities of the
bud and budding zone are less clear: in two experiments the incorporation by
the bud is somewhat higher than that of the budding zone, but in the third
44
S. G. CLARKSON
experiment the situation is reversed to such an extent that the pooled data from
the three experiments indicate greater incorporation by the budding zone than
by the bud.
Table 7. Regional incorporation of [3H]uridine into RNA of
budding hydra
Specific activity (c.p.m.//tg RNA)
Regions
Hypostome and
tentacles
Digestive zone
Budding zone
Bud
Peduncle and
basal disk
%of
maximum
mean
specific
activity
(
Exp. 1
Exp. 2
Exp. 3
Mean
specific
activity
65-4
47-9
35-4
37-6
62-2
450
33-6
34-7
61-7
48-2
461
36-4
631
470
38-4
36-2
100
74
61
57
26-2
29-5
32-8
29-5
47
A
Table 8. Regional incorporation of [uC]algalprotein hydrolysate
into protein of budding hydra
Specific activity (c.p.m .//*g protein)
Exp. 1
Exp. 2
Exp. 3
Mean
specific
activity
431
20-2
20-9
20-9
38-7
22-4
26-3
19-9
43 0
21-8
17-4
23-5
41-6
21-5
21-5
21-4
100
52
52
51
230
17-4
19-5
200
48
K
Regions
Hypostome and
tentacles
Digestive zone
Budding zone
Bud
Peduncle and
basal disk
%of
maximum
mean
specific
activity
(c) Protein synthesis
The procedure in this experiment was exactly the same as that in the previous
one except that [3H]uridine was replaced by [14C]algal protein hydrolysate
(640 /*c/mg) at 7-5 /jc/ml. Results are shown in Table 8.
The only feature common to all three experiments is that the maximum
specific activity is found in the hypostome and tentacles. More proximal regions
vary as to which region possesses the second highest specific activity, but the
extent of this variation is small. When the data from the three experiments are
pooled, the results indicate an approximately uniform incorporation of [14C]algal
protein hydrolysate into all regions proximal to the hypostome and tentacles.
Thus, an axial gradient in incorporation exists only between the hypostome and
Pattern regulation in hydra. I
45
tentacles and the digestive zone, and no major quantitative differences exist
between the more proximal regions.
4. Regional nucleic acid and protein content
The preceding data on the regional patterns of syntheses involved chemical
analyses of the nucleic acid and protein content of the five regions in order to
Table 9. Regional nucleic acid and protein content of budding hydra
Regions
DNA
Og/region)
RNA
Og/region)
Protein
Og/region)
Hypostome and tentacles
Digestive zone
Budding zone
Bud ('medium bud'stage)
Peduncle and basal disk
Total/tg/hydra
0-24
0-38
014
017
009
102
0-86
1-83
0-59
0-82
0-40
4-50
2-64
5-66
2-45
1-89
1-32
13 96
O-10-i
-Mean
c
O)
0073
o
o. 005 <f
Z
Q
O-30-i
Mean
023
^ 0 15-
<T
Z
Q
ein
0-50Mean
032
o
£. 0-25 Z
Bud
Hypostome
and
tentacles
Digestive
zone
I
h
|
I Budding
| zone
I
rl
Peduncle and
basal disk
LJ
Fig. 3. DNA/protein, DNA/RNA, and RNA/protein ratios of budding hydra.
46
S. G. CLARKSON
express radioactivity in terms of specific activities. The results of a typical
analysis are shown in Table 9, where each value for the regional nucleic acid or
protein content is the mean of three determinations, each of which was made on
ten pooled regions. It should be noted that the DNA values were recorded from
2-day starved hydra, whereas the RNA and protein values were obtained from
animals 17-20 h after feeding.
In Fig. 3 are shown the regional DNA/protein, DNA/RNA, and RNA/protein
ratios obtained from the data in Table 9. Some minor chemical differences
between the regions are revealed, e.g. the relatively high DNA/protein and
DNA/RNA ratios of the hypostome and tentacles, and the relatively high
DNA/protein and RNA/protein ratios of the bud. The extent of these differences
is not very large, however, and the DNA/protein ratio of the bud is only 27 %
higher than that of the parent, nowhere near the threefold difference found by
Li&Lenhoff(1961).
5. Nucleic acid and protein synthesis during hypostome formation
(a) DNA synthesis
To determine whether the formation of a hypostome during regeneration is
accompanied by a localized increase in DNA synthesis, use was made of the
specificity of the GSH feeding reflex (Table 1) and the fact that a new hypostome
is determined, in the majority of cases, within 6 h of cutting at the subhypostomal level (Webster & Wolpert, 1966).
Table 10. Incorporation of[3H]thymidine into DNA of intact and
cut subhypostomes incubated in the presence of reduced glutathione
Treatment of subhypostomes
Specific activity
(c.p.m./^g DNA)
Control: Intact 1
230-5]
Subhypo. cut
2
228-8 \
3
261-8J
1
2
3
268-7)
258-3 [
2420j
Mean specific activity
240-4
256-3
Animals were incubated for 6 h in [3H]thymidine (3 c/mM) at 25 jnc/ml in
10~ 5 M GSH in ' M ' . One batch comprised ten intact hydra, and the other, ten
animals cut at the subhypostomal level immediately before incubation. After 6 h
the subhypostomal regions of the two batches were isolated, pooled separately,
and their specific activities determined. Results are shown in Table 10.
A slight increase in mean specific activity is evident in subhypostomes cut
prior to incubation compared to those incubated intact; the difference between
these means is not, however, significant (P > 0-30). These findings confirm the
Pattern regulation in hydra. I
47
earlier experiment on the incorporation by whole animals incubated for 2\ h
(Table 1), and it is clear that incorporation measurements on whole animals do
not mask localized areas of high DNA synthetic activity. Although permeability
changes associated with the feeding reflex probably account for some of the
incorporation obtained by intact animals incubated in the presence of GSH, the
data shown in Tables 4 and 6 indicate that this contribution is relatively small
during a \ h pulse label. Over a 6 h incubation period this effect is considered
negligible because the GSH feeding reflex ceases after approximately 35 min
(Loomis, 1955).
It is therefore concluded that cutting does not stimulate DNA synthesis, and
that during the determination of a hypostome from the subhypostomal region
there is no major increase in DNA synthesis over that occurring in a similar
region left in situ.
(b) RNA synthesis
The procedure in this experiment was exactly the same as that in the previous
one except that [3H]thymidine was replaced by [3H]uridine (3-33 c/mM) at
10//c/ml. Results are shown in Table 11.
Table 11. Incorporation of z[H]uridine into RNA of intact and cut
sub-hypostomes incubated in the presence of reduced glutathione
Treatment of subhypostomes
Specific activity
(c.p.m.//*g RNA)
Control: Intact 1
2
26-7]
33-9 \
3
36-3J
Subhypo. cut 1
2
3
158-3]
164-2 \
132-OJ
Mean specific activity
32-3
151-5
The results demonstrate a highly significant (P > 0-001) increase in [3H]uridine incorporation into subhypostomes cut prior to incubation compared to
those incubated intact. These findings confirm the experiment on the incorporation by whole animals incubated for 2\ h (Table 2), although both are subject
to the criticism that at least part of the increased incorporation following
cutting may be due to the easier entry of the precursor. It is considered unlikely,
however, that such a factor could account for the 370 % increase in incorporation of [3H]uridine into RNA obtained in the present experiment. It is suggested
therefore that cutting does stimulate RNA synthesis, and that during the
determination of a hypostome from the subhypostomal region there is a marked
increase in RNA synthesis compared to that occurring in a similar region left
in situ.
48
S. G. CLARKSON
(c) Protein synthesis
The results of a similar experiment involving the incorporation of [14C]algal
protein hydrolysate (640/*c/mg) at 5/^c/ml into protein of intact and cut
subhypostomes are shown in Table 12.
The present results support the data shown in Table 3 and demonstrate that
cutting at the subhypostomal level leads to a very significant (P < 0-01) 34%
increase in [14C]algal protein hydrolysate incorporation. It is suggested therefore
that there is a significant increase in protein synthesis during the determination
of a hypostome from the subhypostomal region.
Table 12. Incorporation of [uC]algal protein hydrolysate into protein of intact and
cut hypostomes incubated in the presence of reduced glutathione
Treatment of subhypostomes
Specific activity
(c.p.m.//*g protein)
Control: Intact 1
2
420]
40-4 [
3
38-6J
1
2
51-9)
57-3 \
3
52-6J
Subhypo. cut
Mean specific activity
40-3
53-9
DISCUSSION
The experiments have been concerned with nucleic acid and protein synthesis
in intact and regenerating hydra, and the following points are important for the
interpretation of the results.
(1) Radioactivity was determined in total acid-insoluble material so the
possibility cannot be excluded of some incorporation of [3H]thymidine into
RNA, or of [3H]uridine into DNA.
(2) RNA measurements were made without any separation of RNA into its
constituent classes.
(3) The regional patterns of incorporation may not reflect the regional
patterns of syntheses, but rather regional differences in permeability to the
relevant precursor. There is no simple means of resolving this difficulty on the
present evidence since no measurements were made of the specific activity of the
immediate precursor in each of the regions. While permeability is evidently an
important factor in determining the extent of incorporation, as witnessed by the
threefold increase in incorporation of all three precursors when administered in
the presence of reduced glutathione, the similarity in the regional patterns of
[3H]thymidine incorporation obtained after a 24 h incubation and a \ h pulse
label with reduced glutathione indicate, however, that regional permeability
differences are slight.
Pattern regulation in hydra. I
49
(4) No antibiotics were employed and the possibility therefore remains that
an intracellular bacterial population might have significantly contributed to the
radioactivity measurements. This is considered unlikely, however, in view of the
distinctly different regional patterns of incorporation of the three precursors.
Moreover, they are incorporated at very different rates following removal of the
hypostome and tentacles, and it is hardly likely, for example, that bacteria could
account for the large increase in [3H]uridine incorporation under these
conditions. With these possible doubts in mind, it is assumed that the incorporation of each of the three precursors employed in this work is a reasonable
estimate of the relevant synthetic activity in hydra.
The data on the regional pattern of DNA synthesis (Tables 4-6) are in accord
with other quantitative data dealing with the growth pattern of hydra. Mitotic
cells have been found to be generally distributed throughout the body column of
H. littomlis (Campbell, 1967 a; Clarkson & Wolpert, 1967), and this mitotic
distribution is similar to the distribution of [3H]thymidine-labelled nuclei
(Campbell, 1967«). Similar studies on the pattern of mitotic figures in H.
pseudoligactis (Shostak, Patel & Burnett, 1965; Campbell, 19676; H. D. Park,
1967, personal communication), Clytia (Hale, 1964) and Campanularia
(Crowell, Wyttenbach & Suddith, 1965) further indicate that growth is not
restricted to particular zones within hydroids. The present results support this
evidence from histology and radioautography, and it would appear that hydra
possesses neither an axial gradient in DNA synthesis nor any region which can
be termed a localized growth zone.
The suggestion of Ham & Eakin (1958) that a burst of mitosis occurs shortly
after removal of the hypostome and tentacles is not borne out by the experiments
involving reduced glutathione (Tables 1, 10). Rather, they indicate that cutting
does not stimulate DNA synthesis and that during the formation of a new
hypostome from the subhypostomal region there is no major increase in DNA
synthesis over that occurring in an intact subhypostome. This is consistent with
the finding that there is no significant difference between the number of mitotic
figures in intact and 6 h regenerating subhypostomal regions (Webster, 1964).
Moreover, recent histological evidence (Park, Ortmeyer & Blankenbaker, 1967)
indicates that mitotic activity is not increased during any stage of distal regeneration in H. pseudoligactis. It is concluded that the determination of the hypostome
is not accompanied by a localized increase in growth, and the suggestion of
Burnett (1961, 1966) that a new hypostome arises from activation of growth
substances and the formation of a new growth centre is therefore considered
untenable.
The experiments dealing with the regional incorporation of [3H]uridine
(Table 7) indicate a disto-proximal gradient of RNA synthesis, and hence a
possible correlation between metabolic and morphogenetic activities. In
contrast, the data shown in Table 8 are consistent with the concept of an axial
gradient of protein synthesis only in so far as the hypostome and tentacles
4
JEEM2I
50
S. G. CLARKSON
possess greater activity then the remaining body regions. In general terms, these
results bear out the suggestion of Child (1941) that 'high physiological activity'
is associated with a region possessing organizing properties, but of course they
do not establish a causal link between RNA or protein synthesis and the factors
responsible for the formation and localization of the hypostome to the distal end.
The experiments can be interpreted only in terms of total RNA and total
protein synthesis and they indicate that RNA synthesis is more likely to be
important than protein synthesis in the control of polarized regulation in hydra.
Despite the limitations of this level of analysis, it is of interest to consider these
results in relation to the axial variations in the time-threshold and inhibitory
factors proposed by Webster (1966a, b). Since it is assumed that the inhibitor
need only be produced by the hypostome whereas the time-threshold properties
of a region are determined by its position on the linear axis (Webster, 1966 a, b),
it is tempting to relate the time-threshold properties, rather than the level of
inhibition, to the axial variations in RNA synthesis. The assumption that the
threshold of a region is more stable than the level of inhibition (Webster,
1966a, b) would also lend support to this idea. On the other hand, it is far from
clear why such gross regional differences in total RNA synthesis should exist,
and more basic facts are required—for example, the classes of RNA involved—
before definite conclusions can be drawn with regard to the significance of the
axial variations in RNA and protein synthesis.
The experiments in which reduced glutathione was employed to increase
[3H]uridine and [14C]algal protein hydrolysate incorporation suggest that the
formation of a hypostome is accompanied by a very large increase in RNA
synthesis (Tables 2, 11) and a slight but significant increase in protein synthesis
(Tables 3, 12). While reduced glutathione allows an estimate to be made of the
amount of incorporation due to the greater availability of precursor to endodermal cells via the coelenteron, the possibility cannot be excluded that part, if
not all, of the apparent stimulation of RNA and protein synthesis is due to the
precursor entering the cut surface and traversing the mesoglea. However, the
lack of stimulation of [3H]thymidine incorporation under identical conditions
(Tables 1,10) would suggest that precursor entry via the cut surface plays either
a minor or highly variable role in determining the extent of incorporation
obtained after cutting. Of the two, the former is considered more likely and it is
therefore suggested that the apparent stimulation of RNA and protein synthesis
during distal regeneration is genuine.
These results raise some interesting possibilities with regard to the sequence of
metabolic events that occur during hypostome formation. Following a 6 h
incubation of cut subhypostomes, RNA synthesis is increased by 370%
(Table 11), while a 2\ h incubation of whole animals results in a 290 % increase
in [3H]uridine incorporation in the cut samples (Table 2). The RNA content of
the subhypostomal region represents approximately 14% of the total RNA
content of hydra (Table 9). Thus, if the 290 % increase in incorporation obtained
Pattern regulation in hydra. I
51
in the latter experiment is due to a stimulation of RNA synthesis only in the
subhypostomal region, the extent of this stimulation would have to be of the
order of 2100%. While this possibility cannot be entirely eliminated, it seems
more likely that RNA synthesis is stimulated throughout the whole animal, or at
least stimulated not merely within the subhypostomal region, during the first
2\ h of distal regeneration.
In the same way, comparison of the data in Tables 3 and 12 might suggest that
protein synthesis is slightly stimulated throughout the whole animal within 2\ h
of cutting at the subhypostomal level. This suggestion is less easily justified,
however, in view of the small increase in protein synthesis obtained in these
experiments. It should be noted that at least some of the increased incorporation
obtained in the experiments shown in Tables 2 and 3 is likely to be due to the
easier access of the precursors to the explanted hypostomes and tentacles since
both proximal and distal parts were retained for incubation in these experiments.
The low values for RNA and protein content of this region suggest, however,
that such contributions would be relatively small.
The very large increase in RNA synthesis obtained in these experiments
suggests once more that this is the metabolic activity initially and primarily
involved in pattern regulation in hydra. Moreover, the results support the
tentative suggestion that RNA synthesis is likely to be related to the timethreshold properties of a region for, although hypostome formation is accompanied both by the restoration of the level of inhibition to that of intact hydra
and by a rise in the threshold for inhibition, it is assumed that the inhibitor need
only be produced by the hypostome, whereas threshold must rise throughout the
system for regulation to occur (Webster, 1966&). Thus it appears likely that the
stimulation of RNA synthesis within the presumptive hypostomal region
represents in part the synthesis of RNA molecules coding for the inhibitor, while
the apparent rise in RNA synthesis throughout the system is a manifestation of
changes in threshold properties. This is of importance in relation to the possibility that the gradient in threshold could be the factor responsible for the axial
differences in time for hypostome formation, and a question of interest now would
be whether there are qualitative differences in the populations of RNA molecules
synthesized during hypostome formation at different body levels, or whether
there are simply differences in the rates of synthesis of the same populations.
The results reported in this paper provide additional evidence for the earlier
conclusion (Clarkson & Wolpert, 1967) that the factors determining growth in
hydra are quite different from those determining its form. In contrast, both
RNA and protein synthesis appear to have important roles in pattern regulation
in hydra, but it is clear that an understanding of the precise nature of these roles
will require much further investigation. One approach towards this goal is the
experimental alteration of pattern with compounds that primarily affect only
one type of metabolic activity. This will be investigated in the following paper
(Clarkson, 1969).
4-2
52
S. G. CLARKSON
SUMMARY
1. Biochemical techniques were used to determine (a) whether axial gradients
in DNA, RNA or protein synthesis exist in hydra, and (b) whether the formation
of the hypostomal region is accompanied by radical changes in these metabolic
activities. A new technique for administering radioactive precursors was
devised to help answer the second question.
2. [3H]thymidine incorporation is approximately constant within the gastric
region, very similar within the digestive zone, and somewhat lower in the
hypostome and tentacles, and peduncle and basal disk.
3. The regional pattern of [3H]uridine incorporation follows a disto-proximal
gradient with the hypostome and tentacles possessing the highest incorporation.
4. [14C]algal protein hydrolysate incorporation is maximal in the hypostome
and tentacles, but lower and approximately uniform in the remaining body
regions.
5. The formation of a new hypostome appears to be accompanied by no
increase in DNA synthesis, a large increase in RNA synthesis, and a slight
increase in protein synthesis. The stimulation of RNA synthesis probably occurs
throughout the whole animal, and not merely within the presumptive hypostomal
region, during the first 2\ h of distal regeneration.
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. I. La topographie et Vextent des syntheses pendant la
formation de la region hypostomale.
1. Des techniques biochimiques ont ete utilisees pour etudier (a) l'existence
eventuelle de gradients axiaux de synthese de DNA, de RNA ou de proteines,
(b) l'hypothese selon laquelle la formation de la region hypostomale s'accompagne de modifications profondes de l'activite metabolique de cette region. Une
nouvelle technique d'administration de precurseurs radioactifs a ete mise au
point pour repondre a la seconde question.
2. L'incorporation de la 3H-thymidine est quasi constante dans la region
gastrique, tres similaire dans la region digestive et legerement moindre dans la
region de l'hypostome et des tentacules, ainsi que dans celle du pedoncule et du
disque basal.
3. La topographie de l'incorporation de la 3H-uridine revele un gradient
distoproximal; l'incorporation la plus elevee s'effectue au niveau de l'hypostome
et des tentacules.
4. L'incorporation d'un hydrolysat de proteines-14C est maximale au niveau
Pattern regulation in hydra. I
53
de l'hypostome et des tentacules; dans les autres regions, on observe une
incorporation moindre et relativement uniforme.
5. On n'observe pas d'augmentation de la synthese du DNA au moment de la
formation d'un nouvel hypostome; par contre, la synthese du RNA est fortement
augmentee et la synthese proteique legerement accrue. Cette stimulation de la
synthese du RNA affecte non seulement la region hypostomale presomptive,
mais aussi toutes les autres parties de l'hydre, pendant les 190 premieres
minutes de la regeneration.
6. La signification de ces resultats est discutee en fonction des hypotheses
actuelles concernant la regulation de la morphogenese chez l'hydre.
I am deeply indebted to Professor Lewis Wolpert for his advice and encouragement. We
wish to thank the Agricultural Research Council for a scintillation counter, and the Nuffield
Foundation for their support of this work.
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{Manuscript received 30 April 1968)