/ . Embryol. exp. Morph. Vol. 18, 2, pp. 181-97, October 1967
With 1 plate
Printed in Great Britain
181
Studies on pattern regulation in Hydra
IV. The effect of colcemide and puromycin
on polarity and regulation
By GERALD WEBSTER 1
From the Zoology Department, King's College, University of London
INTRODUCTION
Regulation in hydra is polarized, the new hypostome always forming at the
distal end of an isolated piece. Polarity is rigorously maintained in such pieces
but can be altered in graft combination (Peebles, 1900; King, 1901; Morgan,
1901; Browne, 1909; Goetsch, 1929; Tardent, 1960; Webster, 19666).
Previous investigations (Webster, 1966a, b; Webster & Wolpert, 1966) have
suggested that polarized regulation occurs as a result of the interaction of three
factors: time for hypostome determination, inhibition of hypostome determination and threshold for inhibition. All these factors appear to be graded along
the axis. There is some evidence that time for hypostome determination and
threshold for inhibition are closely linked factors (Webster, 19666) and in
many experiments it is not possible to distinguish between them. For this
reason they are referred to throughout this paper as 'time-threshold' factors
or properties.
If polarized regulation does result from interaction between the above
factors, then experimental alteration of polarity would be expected to depend
upon interference with these factors or with their interaction. Experimental
alteration of polarity in graft combinations has previously been discussed in such
terms (Webster, 19666) but no attention has been devoted to experimental
alteration by other means. This is the subject of the present paper.
A large body of literature exists on the effect of various environmental
factors (chemical agents, temperature, electric current, etc.) on regulation and
polarity (see e.g. Child, 1941). In some hydroids, but not hydra, polarity is
normally rather labile and can be influenced by a variety of environmental
factors. Tubularia is a good example (literature reviewed by Child, 1941;
Tardent, 1960, 1963). Corymorpha is a hydroid which often shows bipolar
regulation in short pieces and the frequency of such forms can be increased by
exposing the pieces to inhibitory agents (cyanide, alcohol, low temperature, etc.)
1
Author's address: School of Biological Sciences, University of Sussex, Falmer, Brighton,
Sussex, England.
12
JEEM l8
182
G. WEBSTER
for a short time after isolation. In the majority of such experiments there has
been no detailed analysis of the effect of such agents at either a biological
or a biochemical level. It has usually been assumed that the alteration of polarity
has been produced as a result of interference with some sort of axial gradient
which 'controls' polarity but little convincing evidence has been advanced to
support this view.
It is surprising that little work along these lines has been carried out with
hydra. Lesh & Burnett (1964,1966) have recently demonstrated that a substance
is present in homogenates of H. pirardi and H. viridis which when applied to
isolated pieces of H. pirardi is capable of altering polarity. Lentz (1965), using
slightly different techniques, has made a similar observation. All these workers
have interpreted their results in terms of Burnett's (1961) hypothesis of control
of distal regeneration by' growth stimulating' and' growth inhibiting' substances.
The basis for the experiments described in this paper is the work of Flickinger
(Flickinger, 1959; Flickinger & Coward, 1962), originating from the observations of Kanatani (1958) that brief treatment of planaria with colcemide
(desacetylmethylcolchicine) causes bipolar regulation in isolated pieces. Flickinger showed that there was a gradient in the rate of incorporation of labelled
amino acids into protein along the body of the animal, and demonstrated that
treatment with colcemide or chloramphenicol flattened or abolished this
gradient. Thus the morphological effect of colcemide could be related to a
specific biochemical effect.
In this paper experiments are described on the effect of colcemide on polarized
regulation in various isolated regions of hydra. The effect of this substance on
hypostome formation and the properties of the determined and 'adult' hypostome are also considered. Experiments have also been carried out to determine
the effect of colcemide on those properties (' time-threshold' properties) which
are believed to control polarity. Similar experiments have been carried out
using puromycin, also an inhibitor of protein synthesis (Yarmolinsky &
de la Haba, 1959), since preliminary experiments indicated that chloramphenicol
—effective on planaria—is very toxic to hydra.
It must be emphasized that the primary purpose of the experiments was to
investigate how these chemical agents affect polarity at a biological level, and
to consider the results in terms of concepts of control which have been developed
to explain normal regulation. No attempt is made to discuss the possible
biochemical effects of these substances and no assumptions are made that
they are acting in any particular way at a biochemical level. Such consideration
must await the completion of biochemical investigations which are still in
progress.
Effect of colcemide on Hydra polarity
183
MATERIALS AND METHODS
Hydra littoralis were used for all experiments. Details with regard to selection
of animals, treatment during experiments, etc., have been given in Webster &
Wolpert (1966).
Colcemide (a gift from Ciba Laboratories, Horsham, Sussex) and puromycin
dihydrochloride (Nutritional Biochemicals Corp.) were dissolved in ' M '
solution and animals were treated in large volumes of the solution at 26 °C.
After treatment they were washed three times in ' M \
Different regions of the axis were isolated as follows: digestive zones by
cuts just proximal to the ring of tentacles and just distal to the budding zone;
peduncles by a cut just proximal to the budding zone. Hypostomes and tentacles
or peduncles were removed as in the isolation of the digestive zone. Animals
were allowed to extend maximally before cutting.
Transplantation experiments to investigate hypostomal or subhypostomal
properties were performed as previously described (Webster & Wolpert, 1966;
Webster, 1966 a, b).
EXPERIMENTS AND RESULTS
General effects of colcemide and puromycin
Colcemide (5 x 10~5 M, 20 /«g/ml) inhibits reconstitution of all missing parts.
Hydra placed in colcemide after removal of hypostome and tentacles or peduncle
and basal disc show no signs of reconstituting these missing regions within
48 h. Untreated control animals reconstitute both distal and proximal regions
within 36 h at 26 °C. Colcemide-treated animals, after removal from colcemide,
reconstitute missing regions within 48-120 h as described below.
Hydra treated with colcemide react in a characteristic manner. After 24 h
all animals are extremely swollen and if punctured with a needle deflate like
a pricked balloon, often spewing out masses of loose endoderm cells. Many
animals are spherical at 24 h, but after 48 h most have assumed an irregular
shape.
The polarity of hydra which have had hypostome and tentacles removed is
usually unrecognizable after 24 h in colcemide, but the body wall of spherical
animals often shows a characteristic pattern, being colourless and transparent
at the poles as compared with the equator which is dense, pink and translucent.
In some cases this pattern can be seen to represent the original linear axis, as
indicated by debris characteristic of the basal disc still attached to one pole.
If the hypostome and tentacles are not removed, but the animals are wounded
by a small lateral incision or by the removal of proximal regions, the original
polarity is still recognizable after 24 h, but the tentacles are considerably
reduced in size; after 48 h the tentacles have disappeared completely and
polarity is in general no longer recognizable (Plate 1,fig.1). The body of such
animals often has a characteristic shape; longitudinal depressions appear so
184
G. WEBSTER
that when viewed from one pole the animals have a lobed appearance, the
number of lobes being generally equal to the original number of tentacles
(five or six). Buds at a medium stage of development often complete development while still in colcemide and may separate from the parent.
All colcemide-treated animals rapidly become enclosed in a gelatinous
envelope which becomes thicker during the period in colcemide and after
removal. Microscopic examination reveals this envelope to consist mainly of
cytolysed cells, particularly cnidoblasts with nematocysts in all stages of
differentiation. It is interesting to note that when hydra subsequently reconstitute
tentacles these are entirely or partially deficient in nematocysts in about 50 %
of the animals.
Puromycin is toxic and concentrations of 1-1-1-7 x 10~ 4 M (50-75 ju-g/ml)
were the highest that could be employed. Even at these concentrations a proportion of the animals was killed after 24-48 h treatment. Reconstitution
was not completely inhibited, and after 24 h some animals showed signs of
reconstituting missing peduncles and tentacles.
Puromycin does not produce the striking morphological changes seen in
colcemide-treated animals. Intact hydra placed in 1-1 x 10~^ M puromycin show
signs of tentacle and peduncle regression after 24 h, as do those placed in
colcemide, but even after 48 h these regions have not completely disappeared
and most animals are still axiate with recognizable polarity. Buds often continue
to develop and new ones may arise while the animals are still in puromycin.
After removal from puromycin, tentacle reconstitution occurs fairly quickly—
within 24-48 h—but missing proximal regions are often reconstituted very
slowly, particularly in animals which have been treated for 48 h.
Table 1. Effect of colcemide on hydra possessing hypostome and tentacles
Piece treated
Whole animal (wounded)
Animal minus peduncle
and basal disc
No. of
animals
Time in
colcemide
(h)
20
50
24
48
Number
with
multiple
No. dead
distal
or not
structures reconstituted
0
0
0
1
Effect of colcemide on hydra from which hypostome and tentacles are not removed
Animals which are wounded or have had proximal regions removed (i.e.
basal disc and peduncle) and are treated with colcemide for up to 48 h and
then transferred to ' M ' reconstitute the missing regions and are exclusively
monopolar in form. They possess one distinct group of tentacles, located at
one end of a more or less linear axis (Table 1).
J. Embryo/, exp. Morph., Vol. J8, Part 2
G. WEBSTER
PLATE 1
Effect of colcemide on Hydra polarity
185
Effect of colcemide on hydra from which hypostome and tentacles are removed
prior to treatment
When animals which have had the hypostome and tentacles removed (or
these regions together with peduncle and basal disc) are treated with colcemide
for 24-48 h and are then transferred to ' M', some show signs of tentacle
reconstitution within 48 h. Most of these reconstituting animals possess one
localized group of 4-6 tentacles in one region of a more or less linear axis; these
become typical monopolar forms. The remaining animals, which in general
reconstitute tentacles more slowly, include a significant number which show
not one localized group of tentacles but two or more distinct groups, or alternatively, in a few cases, an apparently random arrangement of isolated tentacles
on an irregularly shaped body. Many of these animals regulate within 48 h.
For example, two adjacent but apparently distinct groups of tentacles may
become confluent and a typical monopolar animal is formed, often with branched
tentacles if the latter have fused at their bases. Alternatively two widely separated
groups of tentacles will each acquire a distal axis and a Y-shaped, V-shaped or
linear bipolar form will result (Plate 1, figs. 2-4). Some animals, particularly
those showing an apparently random tentacle distribution, show little sign of
regulation and retain their irregular appearance for as long as 14 days.
It must be emphasized that in general only distal structures, i.e. tentacles
and distal axes, are involved initially in multiplication. In only eight animals
were multiple basal discs or peduncles observed and careful study indicated
that such forms usually arose when the tentacle group formed in the middle
of a long piece, thus leaving two free ends which became peduncles and basal
discs (Plate 1, fig. 6).
The further fate of animals possessing multiple distal structures is variable.
As noted above, extremely irregular animals usually undergo little change;
PLATE 1
Fig. 1. Intact hydra which have been treated with colcemide for 36 h; they have rounded
up and the tentacles have been resorbed. The axial polarity is no longer visible.
Fig. 2. Y-shaped bipolar, produced by treating a hydra minus hypostome and tentacles with
colcemide for 48 h. A basal disc is present (arrow). The thin and transparent appearance of
the tentacles is due to the absence of nematocysts.
Fig. 3. Linear bipolar produced by treating an isolated digestive zone with colcemide for
24 h. No basal disc is present.
Fig. 4. V-shaped bipolar produced by treating an isolated digestive zone with colcemide
for 24 h. A basal disc has developed at the apex of the V (arrow).
Fig. 5. Hydra with multiple distal structures produced by treating an isolated digestive zone
with colcemide for 48 h. Three groups of tentacles can be seen (arrows).
Fig. 6. Irregular bipolar produced by treating an isolated digestive zone with colcemide
for 48 h. Three basal discs (arrows) are present at the ends of the two shared axes; the two
tentacle groups developed earlier at sites near the middle of an irregularly shaped long
piece.
186
G. WEBSTER
Y-shaped bipolars usually develop a basal disc at the foot of the Y within 48 h
of reconstituting tentacles (Plate 1, fig. 2) and subsequently behave like bipolars
produced by grafting a hypostome into the digestive zone of a host animal, i.e.
the two axes show no sign of separating for up to 14 days. V-shaped or linear
bipolars subsequently form a basal disc at the apex of the V or in the middle
of the linear axis (Plate 1, fig. 4), and in many cases separation of the two axes
occurs within 7-14 days. The behaviour of these forms is analogous with that
of bipolars produced by grafting two hydra together with opposite polarity.
Table 2. Effect of colcemide on hydra from which the hypostome and
tentacles are removed prior to treatment
Piece treated
Animals minus hypostome
and tentacles only
(h)
No. with
multiple
distal
structures
22
24
3(12%)
1
40
0
0
No. of
animals
Time in
colcemide
200
48
—
9(22%)
Untreated controls
Isolated digestive zones
24
48
—
14(28%)
9(45%)
Untreated controls
50
20
200
Isolated peduncles
Untreated controls
20
20
24
—
0
No. dead
or not
reconstituted
0
0
0
0
0
0
2
0
The number of animals showing multiplication of distal structures is affected
by both the size of the treated piece and the duration of colcemide treatment
(Table 2). Treatment of hydra minus hypostome and tentacles for 24 h usually
results in a small number (12 %) of the animals reconstituting multiple distal
structures which nearly all regulate to form Y-shaped bipolars with two distinct
groups of tentacles, two distal axes and a common proximal axis and basal
disc. Increasing the time of treatment of animals minus hypostome and tentacles
to 48 h results in an increase in the number of forms showing multiplication
of distal structures (22 %), but again the majority regulate to form Y-shaped
bipolars. Reducing the size of the piece by employing isolated digestive zones
and treating for 24 h results in an increased number of animals with multiple
distal structures (28 %), and these in general regulate to form V-shaped or
linear rather than Y-shaped bipolars. Treatment of isolated digestive zones for
48 h with colcemide results in greatly retarded tentacle reconstitution; some
animals take up to 5-6 days before producing tentacles, but most animals
eventually do so. More than 40 % of these animals show multiple tentacle
sites and in a few cases they are apparently randomly distributed over an
irregularly shaped body. Regulation to produce distinct distal axes does not
Effect of colcemide on Hydra polarity
187
usually take place in these very irregular forms. Animals which show less
drastic alteration regulate to form V-shaped or linear bipolars.
In contrast to the behaviour of isolated digestive zones, peduncles when
isolated and treated with colcemide for 24 h reconstituted to produce exclusively
monopolar forms.
Effect of colcemide on hydra from which the hypostome and tentacles are removed
subsequent to treatment
When intact animals are treated with colcemide for 24 h, removed from
colcemide and the digestive zone isolated (i.e. hypostome and tentacles, budding
zone, peduncle and basal disc removed) they reconstitute to produce exclusively
monopolar forms (Table 3 (1)). Animals which are treated with colcemide for
24 h after removal of the peduncle and basal disc and then have the hypostome
and tentacles removed subsequent to colcemide treatment behave in a similar
fashion; out of fifty animals treated, only one reconstituted to form a linear
bipolar with a tentacle group at each end of the long axis (Table 3 (2)).
Table 3. Effect of colcemide on hydra from which the hypostome
and tentacles are removed subsequent to treatment
Piece isolated after
treatment
No. of
animals
Time in
colcemide
(h)
Digestive zone (1)
Digestive zone (2)
32
50
24
24
No. with
multiple
No. dead
distal
or not
structures reconstituted
0
1(2%)
5
17
It should be emphasized that there are certain technical difficulties in this
experiment. As described above, animals which have been treated with colcemide
for 24 h are considerably swollen and have partially or totally resorbed their
tentacles. For this reason it is extremely difficult to be certain that all the
hypostome and tentacles have been removed, since these regions are distorted
and spread over a considerable area of one pole of the spherical animal. In an
effort to ensure removal of all of these regions rather large amounts of tissue
were usually cut away and many of the pieces were therefore considerably
smaller in size than in the other experiments. This may account for the fact
that a large number of animals failed to reconstitute tentacles in one of these
experiments which employed a smaller piece (animal minus peduncle and
basal disc) to start with. The single animal showing multiple distal structures
may therefore simply be a result of a failure to remove completely all of the
hypostome and tentacles, in which case the animal would be expected to
reconstitute in a similar manner to those from which the hypostome and tentacles
are not removed subsequent to colcemide treatment.
188
G. WEBSTER
Effect of puromydn on hydra from which the hypostome and tentacles are
removed prior to treatment
Isolated digestive zones when treated for 24-48 h with 1-1 x 10"4 M puromycin
reconstitute to form typical monopolar hydra (Table 4). One animal from the
24 h group produced a secondary distal axis bearing a single tentacle (very
similar to a type 3 induction—Webster & Wolpert, 1966) giving rise to a Y-shaped
bipolar.
Treatment of isolated proximal and distal halves of the digestive zone with
a slightly higher concentration of puromycin (1-7 x 10~ 4 M) resulted in a small
increase in the frequency of bipolar reconstitution (Table 4).
Bipolars produced from the distal half of the digestive zone were all Y-shaped
of exactly the same type as the one described above. The single one produced
from the proximal half was a linear bipolar with two distinct tentacle groups
at opposite ends of the long axis.
Table 4. Effect of puromycin on hydra from which the hypostome and
tentacles are removed prior to treatment
Piece treated
Isolated digestive zone
Distal half of digestive zone
Proximal half of digestive zone
Time in
No. of puromycin
animals
(h)
20
20
20
20
24
48
24
24
No. with
multiple
distal
structures
No. dead
or not reconstituted
1 (5%)
0
4(20%)
1(5%)
1
6
7
17
Thus, although puromycin has similar effects to colcemide in causing treated
animals to reconstitute multiple distal structures, it is definitely not as effective
as the latter substances. Both a higher concentration of chemical and a smaller
size of treated piece are necessary before animals with multiple distal structures
are produced in significant numbers.
Effect of colcemide on hypostome formation and on the organizing properties of
the hypostome
The effect of colcemide on the formation of a new hypostome was tested by
transplanting the distal tip of a reconstituting animal to the digestive zone of
an intact host hydra (Webster & Wolpert, 1966). Animals which had been
reconstituting in colcemide (5 x 10~5 M) for 8-9 h showed no signs of hypostome
formation from the subhypostomal region, though the majority of control
animals possessed determined hypostomes at this time (Table 5).
The organizing ability of the hypostome was unaffected by colcemide treat
ment. Fragments of hypostome taken from hydra which had been treated (in
Effect of colcemide on Hydra polarity
189
the absence of peduncle and basal disc) for 16-17 h induced secondary axes
in the majority of the hosts to which they were transplanted (Table 5). The
morphological changes and the consequent obliteration of visible polarity
which result from colcemide treatment prevent this experiment being performed
on animals which have been treated for longer periods.
Table 5. Effect of colcemide on the formation and organizing
properties of the hypostome
Time after
cutting when
grafted
Time in
colcemide
Source of graft
(h)
(h)
Subhypostome
8-9
8-9
—
8-9
—
Hypostome
16-17
No. of
successful
grafts
No. of
animals
with
secondary
axis
18
10
19
0
8
17
Effect of colcemide and puromycin on the determined hypostome
Subhypostomal regions were isolated and allowed to reconstitute in ' M '
for 8 h. At this time, transplantation of pieces to the digestive zone of intact
host hydra indicated that a determined hypostome was present in the majority
of cases (Table 6). The remaining pieces were transferred to colcemide (5 x
10~5 M) or puromycin (1-1 x 10~4 M) for 16 h, when transplantation experiments
were again performed. Treatment for this time in either substance does not
result in irreversible changes or death; washed pieces transferred to ' M ' all
reconstituted tentacles within 24-48 h. It is important to note that about 50 %
of the pieces which had been treated with puromycin possessed small tentacle
buds at the time of transplantation—clear evidence of the presence of a functional
hypostome.
The results shown in Table 6 indicate that colcemide treatment has no effect
on the determined hypostome, which induced secondary axes in the majority
of cases. This confirms the results previously obtained with the 'adult' hypostome. Pieces treated with puromycin, however, induce secondary axes in a
very few cases only, the majority of the grafts being absorbed. This indicates
that puromycin treatment interferes with the ability of the determined hypostome to resist the influence of the factors which bring about absorption
following transplantation (Webster, 19666). It is interesting that one puromycintreated graft which did not induce was not absorbed, but transformed into
a small peduncle complete with basal disc, a striking indication of a change
in properties to those of more proximal regions.
190
G. WEBSTER
Effect of colcemide and puromycin on the properties of the subhypostomal region
Freshly isolated subhypostomal regions were treated with colcemide (5 x
10~5 M) or puromycin ( I - I X I O ^ M ) for 9 h and then transplanted to the
digestive zones of host hydra from which the hypostome and tentacles had
been removed—the usual test for subhypostomal 'time-threshold' properties
(Webster, 1966#, b). Treatment for this time does not produce irreversible
changes or injury; washed pieces transferred to ' M ' all reconstituted tentacles
within 24 h.
Table 6. Effect of colcemide and puromycin on the determined hypostome
Treatment
None
Colcemide
Puromycin
Time after
Period when cutting when
No. of
treated
grafted
successful
(h)
(h)
grafts
—
8-24
8-24
8
24
24
15
20
20
No. of
animals with
secondary
axes
10(66%)
16(80%)
3(15%)
Table 7. Effect of colcemide and puromycin on the properties
of the subhypostomal region
Treatment
None
Colcemide
Puromycin
Time after
Period when cutting when No. of
treated
grafted
successful
(h)
(h)
grafts
—
0-9
0-9
0
9
9
10
18
20
No. of
animals with
secondary
axes
6(60%)
1 (6%)
0
Puromycin treatment resulted in all the grafts being absorbed. In the case of
grafts treated with colcemide, only one case of induction of a secondary axis
was observed. Control grafts (untreated) made immediately after isolation
behaved in the usual way and produced secondary axes in the majority of
cases (Table 7).
The results indicate that treatment of the subhypostomal region with colcemide or puromycin changes the 'time-threshold' properties of this region to
those characteristic of more proximal regions (Webster, 1966a, b). The results
also, of course, confirm that colcemide inhibits hypostome formation. Whether
puromycin acts in a similar fashion cannot be determined since, as the previous
experiment showed, puromycin intereferes with the resistance of the hypostome
to factors bringing about absorption.
Effect of colcemide on Hydra polarity
191
DISCUSSION
The results show that colcemide treatment of hydra from which the hypostome
has been removed can bring about an alteration of polarity such that multiple
distal structures, i.e. more than one hypostome, can form in a single animal.
The alterations observed were variable and resulted in the production of a range
of forms, from animals showing bipolar organization to those in which hypostomes were more or less randomly distributed. Pretreatment with colcemide
followed by removal of the hypostome resulted in only one animal reconstituting
more than one hypostome. In no case did such forms arise if the original
hypostome was not removed. Treatment with puromycin in the absence of the
hypostome was much less effective in altering polarity as judged by subsequent
reconstitution than was treatment with colcemide. The effects of colcemide and
puromycin on 'physiological' polarity were paralleled by their morphological
effects; visible polarity was completely obliterated in colcemide treated animals
but persisted in those treated with puromycin.
The transplantation experiments indicated that hypostome formation was
inhibited by colcemide but that this substance did not irreversibly affect the
determined hypostome or the 'adult' hypostome, which retained their organizing properties. The results of puromycin treatment are curious. Hypostome
formation was not inhibited completely, as indicated by the fact that some
animals reconstituted tentacles while in puromycin. However, the hypostome
which was formed did not possess the normal resistance to absorption, as
shown by the transplantation experiments. Both colcemide and puromycin
changed the ' time-threshold' properties of the subhypostomal region to those
characteristic of more proximal regions.
The simplest way of explaining the effects of colcemide and puromycin on
regulation is to postulate that treatment with these substances flattens or
abolishes the disto-proximal axial gradient in 'time-threshold' properties
(Webster, 1966 a), so that when released from inhibition distal regions have no
advantage over proximal regions as regards hypostome formation. The results
of the experiments on the subhypostomal region are direct evidence that treatment with either substance changes the 'time-threshold' properties of this region
to those characteristic of more proximal regions, i.e. regions which are lower on
the gradient. In order that the gradient be flattened, it is clear that distal regions
must be affected more than proximal regions. No evidence is available on this
point, though the fact that colcemide treatment of isolated peduncles did not
result in the formation of multiple distal structures may indicate that proximal
regions are less affected. This is also suggested by the fact that animals from
which the hypostome and tentacles only have been removed (i.e. animals possessing a peduncle) produced mainly Y-shaped bipolars with a hypostome at each
end of the digestive zone. Kanatani (1958) observed that in planaria bipolar
reconstitution was more frequent from anterior than from posterior regions.
192
G. WEBSTER
Consider what would happen when an animal whose hypostome has been
removed is placed in colcemide. New hypostome formation is prevented and
the level of inhibition will fall, presumably throughout the animal. The gradient
in' time-threshold' properties is flattened. On removal from colcemide, all regions
will begin forming hypostome, and those with the 'highest' 'time-threshold'
properties will succeed and inhibit those with 'lower' properties. The final
result will be variable and animals will form one or more hypostomes, depending
on how the gradient has been altered and how the developing hypostomes
influence each other. A hypostome, once formed, will immediately begin to
raise the level of inhibition in its immediate neighbourhood (Webster, 19666).
It is probable that any other hypostome which forms will be as far away from
the first as possible, since we know that the effectiveness of inhibition decreases
with distance (Webster, 1966a). For example, in an isolated digestive zone the
new hypostomes will form at opposite ends and a linear bipolar will result.
When the original hypostome was left in place during colcemide treatment
and subsequently removed, multiplication of distal structures was observed in
only one animal out of those which reconstituted. It will be remembered that
there were technical difficulties in this experiment, and the possibility that the
hypostome was not completely removed in all animals makes it unwise to
conclude that this procedure is less effective in altering polarity. However, it is
possible that leaving the hypostome in situ during treatment tends to counteract
the action of colcemide, and this is consistent with the idea that the hypostome
plays some role in controlling the gradient in 'time-threshold' properties
(Webster, 19666).
In those experiments in which the hypostome was left in place both during
and subsequent to colcemide treatment no multipolar forms were produced.
Even if the gradient is flattened in these cases, we have seen that the determined
hypostome is not irreversibly affected by colcemide treatment, so that, on
removal from the substance, a functional hypostome is present which can
presumably inhibit hypostome formation elsewhere in the animal. Even if the
inhibitory action of the hypostome is impaired by colcemide treatment, suppression of new hypostome formation will occur at a low level of inhibition
because of the depression of thresholds.
Puromycin is much less effective than colcemide in causing the production
of multipolar forms, even though it has a similar effect to the latter substance
on 'time-threshold' properties. This is probably due to the fact that puromycin
does not inhibit the formation of a hypostome in all animals. An animal
(minus hypostome) when placed in puromycin may develop a new hypostome
which, although abnormal in resistance to absorption when transplanted, is
adequate to inhibit further hypostome formation in situ. Thus reconstitution
will be monopolar.
It is interesting to note that colcemide treatment produced about twice as
many multipolar forms from isolated digestive zones as compared with larger
Effect of colcemide on Hydra polarity
193
pieces (animals minus hypostome and tentacles only). Similar results were
obtained with puromycin. This seems to be a general feature of bipolar reconstitution. For example, in marine hydroids the frequency of spontaneous
bipolar forms is higher in small pieces than in large pieces (Child, 1941).
A completely satisfactory explanation of this result is not at present possible. It
is clear that the two ends of a small piece will have fairly similar' time-threshold'
properties so that it is easier for them to become identical than for the two
ends of a large piece. However, this explanation will not account for the difference
in numbers between small and large pieces, unless the additional assumption
is made that a hypostome has a greater tendency to arise from a cut surface
(i.e. an end) than from an intact area.
Although colcemide and puromycin have similar effects on the' time-threshold'
properties of the subhypostomal region, their effects on hypostome formation
and the determined hypostome are strikingly different. Colcemide appears to
block hypostome formation completely, but the determined hypostome is
resistant to and is not irreversibly affected by colcemide treatment. The behaviour
of the determined hypostome with respect to colcemide is reminiscent of its
behaviour with respect to the 'natural' inhibitor of hypostome formation
(Webster, 1966a).
The effect of puromycin is extremely interesting. This substance does not
appear to inhibit hypostome formation since organizing properties may be
acquired in some cases (as judged by the production of tentacles). However, it
seems to interfere with the resistance to absorption of the determined hypostome,
and presumably with the acquisition of resistance during hypostome formation.
This suggests very strongly that organizing ability and resistance to absorption
are quite distinct properties. If the resistance to absorption normally displayed
by the determined hypostome is dependent on resistance to inhibition and
therefore upon high threshold, the fact that organizing properties can be
acquired and retained in the absence of resistance suggests that the observed
rise in threshold during hypostome formation (Webster, 1966 b) might not be
a necessary part of the process of hypostome formation. This has important
consequences for our understanding of how release from inhibition occurs
during normal regulation. If hypostome formation can occur without rise in
threshold, then release from inhibition must result from a fall in the level of
inhibition.
If the resistance to absorption displayed by the hypostome is dependent on
threshold, then its threshold properties must be less labile than those of nonhypostomal regions since we have seen that subhypostomal threshold properties
are altered by both colcemide and puromycin but hypostomal resistance is
unaffected by colcemide. It is, of course, possible that higher concentrations
of colcemide might interfere with hypostomal resistance. It is known that the
threshold properties of non-hypostomal regions are unstable and change as
a consequence of isolation or transplantation. The hypostome, however, retains
194
G. WEBSTER
its characteristic properties following transplantation and for as long as 7 days
after isolation (Webster, 19666, and unpublished observations).
An alternative explanation is possible for the different results obtained with
colcemide and puromycin if it is assumed that colcemide has no direct effect
upon 'time-threshold' properties and that these change as a result of the inhibition
of hypostome formation. It is known that the 'time-threshold' properties of
a region are determined by the position of the region on the linear axis in
relation to the hypostome. Changing the position of a region so that it is
nearer the hypostome 'raises' the 'time-threshold' properties; moving it farther
away from the hypostome' lowers' the' time-threshold' properties. These changes
only occur if the level of inhibition remains above the threshold so that hypostome formation is prevented (Webster, 19666). It seems reasonable to postulate
that if a region is removed to an 'indefinite' distance from a hypostome, i.e.
isolated, and at the same time prevented from forming hypostome by colcemide,
then 'time-threshold' properties would 'fall'. This explanation is consistent with
the observations that colcemide has little effect on the polarity of animals
treated prior to hypostome removal and that the determined hypostome is
resistant to treatment. Puromycin, on the other hand, would be supposed to
have a direct effect on 'time-threshold' properties and hence could affect both
hypostomal and non-hypostomal regions. Its relative inefficiency at bringing
about alteration of polarity would result from its failure to inhibit hypostome
determination. This explanation could be subjected to experimental test by
examining the 'time-threshold' properties of regions of animals treated with
colcemide in the presence of the hypostome. It might be mentioned that in
planaria polarity can be altered in animals treated prior to head removal
(Kanatani, 1958; Flickinger & Coward, 1962).
The present results are of interest in relation to those of Lesh & Burnett
(1964, 1966) and Lentz (1965). These workers demonstrated that a substance
was present in homogenates of hydra which when applied to isolated pieces
of the digestive zone for 4 h was capable of altering polarity and producing
animals with multiple distal structures. Lesh & Burnett have claimed that this
substance is identical with the so-called 'growth-stimulating principle', postulated by Burnett (1961) to account for distal regeneration in hydra, and also
that the substance is 'responsible for polarity'. The forms produced as a result
of treatment with the homogenate appear to be identical with those produced
in the present experiments as a result of colcemide treatment. Colcemide is an
inhibitor of protein synthesis in planaria (Flickinger, 1959) and of mitosis in
a variety of organisms (Schar, Loustalot & Gross, 1954) including hydra
(Webster, 1964); it can therefore fairly be described as a 'growth-inhibiting
substance'. The fact that it appears to have identical effects to the substance
present in hydra homogenates suggests that the conclusion that the latter is
a 'growth-stimulating substance' is unjustified and also suggests that 'growth'
plays no role in the control of polarity. More important, colcemide inhibits
Effect of colcemide on Hydra polarity
195
hypostome formation and the alterations of polarity which are produced
(involving effects on regional 'time-threshold' properties) take place in the
absence of a hypostome. In other words, hypostome formation as such is not
a necessary feature of any alteration in polarity but merely reveals at a later
stage that such alterations have taken place. In the experiments of Lesh and
Burnett alteration of polarity is said to occur as a result of the actual stimulation
of hypostome and tentacle formation. It is worth noting that these workers
treated isolated pieces for only 4 h. If the substance they are dealing with is
really 'growth-stimulating' then continuous treatment during regeneration
should have the same or a greater effect than brief exposure. Also, the substance
should be effective irrespective of the presence or absence of the hypostome.
These critical experiments do not appear to have been done. In the light of these
criticisms it must be concluded that the significance for normal regulation of
the substance present in hydra homogenates is obscure.
SUMMARY
1. The effect of colcemide and puromycin on polarity and regulation in
hydra has been investigated at the biological level using isolation and transplantation techniques.
2. Colcemide treatment of hydra from which the hypostome has been removed
can bring about an alteration of polarity so that multiple distal structures
(hypostome and tentacles) can form on a single animal. Such forms do not
arise if the original hypostome is not removed. Treatment with puromycin is
much less effective in altering polarity.
3. Transplantation experiments indicate that hypostome formation is inhibited by colcemide treatment but that this substance does not irreversibly
affect the determined hypostome or the 'adult' hypostome which retain their
organizing properties.
4. Puromycin treatment does not inhibit hypostome formation completely
since some animals reconstitute tentacles while in puromycin. However, the
hypostome which is formed does not possess the normal resistance to absorption
following transplantation. This result may indicate that the factors responsible
for the organizing properties of the hypostome (i.e. tentacle induction) are
distinct from those which control resistance to absorption.
5. Both colcemide and puromycin change certain important properties of
the subhypostomal region to those characteristic of more proximal regions.
These properties are believed to 'control' polarity.
6. The effects of colcemide and puromycin on polarity and regulation are
discussed in terms of their effects on a control system which has been previously
postulated to account for polarised regulation in hydra. This system involves
axial gradients in time for hypostome determination, inhibition of hypostome
formation and threshold for inhibition. The experimental results can be explained
as a consequence of the direct or indirect action of these substances on the
axial gradients.
196
G. WEBSTER
RESUME
Etudes sur la regulation chez VHydre. IV. Action de la colcemide
et de la puromycine sur la polarite et la regulation
1. L'action de la colcemide et de la puromycine sur la polarite et la regulation
chez l'hydre a ete etudiee au niveau biologique au moyen de techniques
d'isolement et de transplantation.
2. Le traitement a la colcemide d'une hydre dont on a coupe l'hypostome
peut determiner un changement de polarite tel que de multiples structures
distales (hypostome et tentacules) peuvent se former sur un seul animal. De telles
formations n'apparaissent pas si l'hypostome primitif n'est pas supprime. Le
traitement a la puromycine affecte beaucoup moins la polarite.
3. Des experiences de transplantation montrent que la formation de l'hypostome est inhibee par un traitement a la colcemide mais que cette substance
n'affecte pas irreversiblement l'hypostome determine ou l'hypostome 'adulte'
qui conservent leurs proprietes organisatrices.
4. Le traitement a la puromycine n'inhibe pas completement la formation
de l'hypostome puisque quelques animaux reconstituent des tentacules pendant
ce traitement. Cependant, l'hypostome qui est forme ne possede pas la resistance
normale a l'absorption qui suit la transplantation. Ce resultat peut indiquer
que les facteurs responsables des proprietes organisatrices de l'hypostome
(c'est-a-dire 1'induction des tentacules) sont distincts de ceux qui controlent la
resistance a l'absorption.
5. La colcemide et la puromycine transforment certaines proprietes importantes de la region sous-hypostomale en proprietes caracteristiques des regions
plus proximales. On pense que ces proprietes 'controlent' la polarite.
6. Les effets de la colcemide et de la puromycine sur la polarite et la regulation
sont discutes en fonction de leur action sur un systeme de controle qui a ete
precedemment postule pour expliquer la regulation polarisee chez l'hydre.
Ce systeme implique des gradients axiaux dans le temps pour la determination
de l'hypostome, l'inhibition de la formation de l'hypostome et le seuil de
l'inhibition. Les resultats experimentaux peuvent etre interpreted comme une
consequence de l'action directe ou indirecte de ces substances sur les gradients
axiaux.
I should like to thank Dr L. Wolpert for helpful discussions during the course of this
work, and the Agricultural Research Council for a Postgraduate Research Studentship.
Effect of colcemide on Hydra polarity
197
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(Manuscript received 6 January 1967)
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