J. Embryol. exp. Morph. Vol. 16, l,pp. 123-41, August 1966
Printed in Great Britain
123
Studies on pattern regulation in hydra
III. Dynamic aspects of factors controlling hypostome formation
By GERALD WEBSTER 1
From the Zoology Department, King's College, University of London
INTRODUCTION
In the previous papers it was shown that three factors are involved in tne
regulation of hydra into a two-compartment pattern comprising hypostome and
non-hypostomal regions (Webster & Wolpert, 1966 a; Webster, 1966). These
three factors are time for hypostome determination, level of inhibition, and
threshold for inhibition. All these factors appear to be graded along the axis.
The interaction between the three factors was shown to be capable of accounting
for polarized regulation, but it was pointed out that a more detailed explanation
would necessitate knowledge of the dynamics of these factors during regulation.
In particular it is important to know how threshold and level of inhibition
change during regulation, and how the gradients themselves are controlled.
Such data is also required to enable an explanation for a variety of grafting
experiments to be offered.
MATERIALS AND METHODS
The hydra used throughout this work was Hydra littoralis. Full details of
experimental methods have been given in Webster & Wolpert (1966 a).
EXPERIMENTS AND RESULTS
Experiment 1. Hypostome formation by a subhypostomal region
transplanted to the digestive zone of a regenerating hydra
The object of this experiment was to determine the time-course of the restoration of inhibition during regulation. The experimental procedure was exactly
as in exp. 1 of Webster (1966) except that regenerating hydra were used as host
animals.
Hosts had hypostomes and tentacles removed and, at various times after
cutting, freshly isolated subhypostomal regions were transplanted to the middigestive zone of these regenerating hydra. The number of animals producing
induced secondary axes is shown in Table 1.
1
Author's address: Strangeways Research Laboratory, Wort's Causeway, Cambridge,
England.
124
G. WEBSTER
It can be seen that subhypostomal regions transplanted to hydra 2-3 h after
the removal of the host hypostome and tentacles behave as do pieces transplanted at the same time as the hypostome is removed (cf. exp. 1, Webster, 1966)
and form hypostomes in the majority of cases. After 5-6 h of distal regeneration
the number of pieces forming hypostome is slightly reduced (not significantly
by x2 test), but after 9 h none of the transplanted pieces induce secondary axes.
Thus an animal which has been regenerating for 9 h is capable of preventing
hypostome formation from transplanted subhypostomal regions in exactly
the same way as an intact hydra (cf. exp. 1, Webster, 1966).
Table 1. Hypostome formation by a subhypostomal region
transplanted to the digestive zone of a regenerating host
Time after
hypostome
removed
when
grafted
(h)
No. of
successful grafts
No. of
animals
with
secondary
distal axes
2-3
5-6
9
20
20
20
14(70%)
10(50%)
0
Discussion of results of Experiment 1
In exp. 1 of Webster (1966), transplantation of subhypostomal regions to the
digestive zones of hydra minus the hypostome resulted in 70 % of the grafts
forming hypostomes; in the presence of the host hypostome, all the grafted
pieces were inhibited. The present experiment gives some indication of how
quickly the inhibitory situation is restored to normal in animals from which the
hypostome has been removed. To be more exact, the results show how quickly
inhibition in the digestive zone is restored to a level above the threshold of the
subhypostomal region.
Although 9 h regeneration is required before all the transplanted pieces are
inhibited, it can be estimated that 50 % of the grafts are inhibited after about
5 h, i.e. 50 % behave as though transplanted to hydra possessing a hypostome.
This time is practically identical with the T50 for hypostome determination from
the subhypostomal region (4-5 h) derived from the results of exp. 1 of Webster &
Wolpert (1966 a), which is very encouraging in view of the different method used.
The fact that restoration of inhibition seems to proceed at the same rate as
hypostome formation indicates that either the level is raised during the formation of the hypostome or almost immediately after the hypostome is determined.
On the present data it is not possible to decide between these alternatives. It
should be noted that there is a margin of 4-5 h before the transplanted subhypostomal region becomes determined as a hypostome and resistant to inhibi-
Pattern regulation in hydra. HI
125
tion. This means that the maximum time for restoration of inhibition in the
digestive zone to a level above the threshold of the subhypostomal region in
50% of the animals is 9-5 h (time of grafting when 50% inhibited+ 4-5 h
margin). Thus, although the level of inhibition may be raised immediately after
the hypostome is determined, the results only allow us to conclude that the level
must be raised within 4-5 h of this event.
The results imply that in the absence of the hypostome, where new hypostome
formation occurs from the subhypostomal region, all regions with a T50 of more
than 9-5 h will be prevented from forming hypostome (in 50 % of the animals)
even if they are initially released from inhibition, since by this time the level of
inhibition in the digestive zone is effectively equivalent to that in the intact
animal. A T50 of 9-5 h defines the most proximal region which can form hypostome under these conditions. However, when the effect of thresholds is taken
into consideration, the proximal limit may become even more restricted, since
9-5 h is the time for restoration of the level of inhibition above the threshold of
the subhypostomal region, i.e. the maximum threshold. It is conceivable that the
level of inhibition rises above the thresholds of other regions in less than 9-5 h,
and if this is so the regions will be inhibited in a proximo-distal sequence, i.e. in
order of increasing threshold.
Experiment 2. Changes in regional threshold properties
during hypostome formation
The isolated peduncle was chosen as the region in which to study the changes
in properties during hypostome formation, since, with a r 50 of 16-5 h (exp. 1
of Webster & Wolpert, 1966 a), there is a reasonable time for experimental
investigation during the process. The object of the experiment was to determine
whether, during hypostome formation, the distal tip of the peduncle passes
through a stage when its properties are comparable to those of a subhypostomal
region, i.e. whether when it has a T6Q equivalent to that of the subhypostomal
region it will behave in other respects like this region.
The experimental procedure made use of the observation in exps. 1 and 3 of
Webster (1966) that a subhypostomal region grafted to the digestive zone in the
absence of the host hypostome would form a hypostome and induce a secondary
axis. The experiment was carried out as follows: peduncles were isolated and
allowed to regenerate in the usual way; 12 h after isolation (r 50 for peduncles
- r 50 for subhypostomal region) the distal tip was removed and transplanted
(1) to the digestive zones of host hydra from which the hypostome and tentacles
were removed, (2) to the basal discs of intact host hydra, (3) to the digestive
zones of intact hydra. The last group were the controls to ensure that no
peduncles had formed determined hypostomes at the time. Results are shown
in Table 2.
It is apparent that after 12 h in isolation the distal tip of the peduncle has
acquired properties comparable with those of the freshly isolated subhypostomal
126
G. WEBSTER
region—it is absorbed in the digestive zone when the host hypostome is present,
but forms a hypostome when it is absent; in the basal disc it forms a hypostome
in a significant number of cases even when the host hypostome is present.
Table 2. Changes in regional threshold properties during hypostome formation
Distal tip
transplanted to
Digestive zone
Basal disc
Digestive zone
Host
hypostome
No. of
successful
grafts
No. of animals
with secondary
distal axes
Absent
Present
Present
17
20
10
11(65%)
8 (40 %)
0
Discussion of results of Experiment 2
The results show that 12 h after isolation of the peduncle, the distal tip of this
region has acquired threshold properties comparable to those of the subhypostomal region. None of the pieces has formed a hypostome at this time.
The results of the experiment involving transplantation to the digestive zone
in the absence of the hypostome are not conclusive with regard to threshold
changes when considered alone. Similar results would have been obtained if,
following the removal of the host hypostome, the level of inhibition in the
digestive zone fell below the threshold for the distal peduncle and was not
restored within 4-5 h, i.e. before a hypostome was determined in the transplanted piece. The results of Exp. 1 suggested that inhibition may not be
restored to a level above the maximum (subhypostomal) threshold until 9; 5 h
after hypostomal removal. The positive results in the case of transplantation to
the basal disc in the presence of the hypostome make this explanation unlikely,
for in this case inhibiting conditions can be regarded as constant and yet
typical subhypostomal behaviour was observed.
At 16-5 h 50 % of the distal peduncle pieces will form a determined hypostome. At 12 h, therefore, the T50 of this region is 4-5 h, which is equivalent to
that of the normal subhypostomal region, and the transplantation experiments
indicate that at this time the threshold properties of the region are also equivalent to those of the subhypostomal region. This experiment provides clear
evidence that the formation of a hypostome from a proximal region involves an
increase in threshold, and that during hypostome formation a region temporarily
acquires the properties of more distal regions before acquiring hypostomal
properties.
The results imply that the factors governing the time for hypostome formation
and the threshold for inhibition are closely linked, or even identical, since a
change in the time properties of a region is accompanied by an appropriate
change in the threshold properties. This is clearly a most important conclusion.
If, during the formation of a hypostome, the threshold of a region rises, it is
Pattern regulation in hydra. Ill
127
clear that the ability to resist inhibition and subsequent absorption will increase
also. Whether the hypostome, which has 'absolute resistance' to absorption, is
simply the region with the maximum threshold is not clear, but this is a possibility which must be kept in mind.
The observed change in threshold of a region which is forming a hypostome
immediately raises the question as to whether similar changes occur in other
regions in the absence of the hypostome. The next experiment is an attempt to
answer this question.
Experiment 3. The formation of a new subhypostomal
region during regeneration
As in the last experiment, and for the same reason, the isolated peduncle was
chosen to study the formation of a new subhypostomal region during distal
regeneration. The question posed in this experiment was: does the new subhypostomal region form at the same time as the new hypostome is determined?
The most unsatisfactory aspect of this experiment, which makes the results
of dubious significance, is the difficulty of identifying that part of the axis which
is the new subhypostomal region at the time when the new hypostome has only
just formed. At this time, of course, there are no tentacles to indicate the
proximal extent of the hypostome so that selection of a piece for transplantation
as a suspected subhypostomal region is largely a matter of guesswork.
Isolated peduncles were allowed to regenerate for 18 h (r 50 for hypostome
determination in peduncle+ 1-5 h) and at this time donor peduncles were
selected. In many, but not all, regenerating peduncles, the distal tip becomes
distinctly more dense and opaque than the remainder of the region due to the
accumulation of endodermal cells. These peduncles were chosen as donors and
the distinct distal tip, but no more, removed as the hypostomal region. A piece
of appropriate size was then removed from the distal end of the remaining
peduncle with the hope that at least part of the subhypostomal region was
included (a 'subdistal' piece) and tested for characteristic subhypostomal
properties by: (1) transplantation to the digestive zones of host hydra minus
hypostomes and tentacles (the majority of pieces), and (2) transplantation to the
basal disc of intact host hydra (a small number only). Some of the distal tips
were transplanted to the digestive zones of intact hydra as controls to ensure
that a determined hypostome was present at this time.
Some peduncles were kept for 36 h, when the new subhypostomal region
could be easily identified since tentacle buds were present. A number of subhypostomal regions from these animals were transplanted to the digestive zones
of hosts minus hypostome and tentacles. Results are shown in Table 3.
The results indicate that 18 h after isolation of the peduncle the 'subdistal'
region, in the majority of cases, does not possess characteristic subhypostomal
properties though a determined hypostome is present in the distal tip. At 36 h,
however, the easily identifiable subhypostomal region possesses characteristic
128
G. WEBSTER
properties in nearly half the animals tested. The difference between the proportions of animals producing secondary axes at 18 and 36 h is probably
significant (x2 test, P < 0-05).
One of the secondary axes produced as a result of transplanting a ' subdistal'
piece to the digestive zone was absorbed within 48 h of developing, as indicated
in the table. This was a perfectly normal looking type 1 axis (Webster & Wolpert,
1966 a), and was the only case ever observed of an axis of this type being
absorbed; it is indicated in the percentage totals since it is clearly a positive
result as far as the subhypostomal properties of the region are concerned.
Table 3. The formation of a new subhypostomal region during regeneration
Source of
graft
'Subdistal'
Distal tip
Subhypostomal
Time
after
peduncle
isolation
No. of
No. of
animals
with
secondary
successful
distal
GO
Transplanted to
grafts
axes
18
18
18
36
Digestive zone-hypostome
Basal disc + hypostome
Digestive zone + hypostome
Digestive zone — hypostome
20
5
10
9
1 (+l*)(10%)
0
7 (70%)
4 (44%)
* Axis completely absorbed 48 h after developing.
Discussion of results of Experiment 3
At a time when the distal tip of the peduncle has acquired hypostomal properties in the majority of cases, it has not proved possible to detect subhypostomal properties in the ' subdistal' region. The difficulties of this experiment
have already been pointed out and it is clearly not possible to draw any but the
most tentative conclusion from the results.
The failure to detect subhypostomal properties in the majority of cases may
quite simply be due to removal of this region with the hypostome since the
proximal extent of the latter is unknown. This possibility cannot be ruled out,
but two facts suggest that it may be an unlikely explanation. First, every effort
was made to ensure that only the minimum amount of axis was removed as the
hypostome. The size of the region removed was closely comparable with the
size of the hypostome (the region distal to the tentacles) in peduncles which had
regenerated for 36 h. Secondly, the region removed was more or less morphologically distinct from the remainder of the axis, although whether this distinction bears any relation to the hypostomal properties of the region is not clear
since it does not occur in all animals.
The alternative explanation for the results is that, the 'subdistal' region has
not acquired hypostomal properties at this time; there has been no increase in
threshold comparable to that which occurs in the region which will become
Pattern regulation in hydra. Ill
129
hypostome. The two positive results could be due to a failure to remove all the
hypostomal region.
At 36 h, when the subhypostomal region can be clearly distinguished, characteristic behaviour was observed in nearly half of the cases. The rather small
number of positive results in this experiment might be an indication that
typical subhypostomal properties were only just being acquired, i.e. after 20 h
in contact with a hypostome. However, in view of the small number of experiments and the absence of control grafts of 'normal' subhypostomal regions,
other explanations are possible.
It is clear that a good deal more experimental work will be necessary before
any definite conclusions can be drawn about changes in threshold properties in
non-hypostomal regions. The available evidence suggests that changes do not
occur in the absence of the hypostome—that there is no spontaneous rise in
threshold comparable to that which occurs in a region forming hypostome. The
fact that threshold changes can be detected after a period of contact with a
hypostome, suggests that they may be induced by this region. These suggestions
must be regarded as extremely tentative.
Experiment 4. The induction of a new subhypostomal
region by a hypostome
The object of this experiment was to determine whether the subhypostomal
region of a secondary axis induced by a hypostome possessed typical subhypostomal properties.
Fragments of hypostome from the region at the base of the tentacles were
transplanted to the digestive zones of intact host hydra. After 24 h these grafts
had in the majority of cases induced a secondary axis and tentacles, the axis
at this time being very short indeed. In one group of animals the hypostome and
Table 4. Induction of a new subhypostomal region by a hypostome
No. of animals possessing
Hypostome
removed from
Primary and secondary
axes
Secondary axis only
No. of
animals
Primary
and
secondary
axes
Primary
axis
only
Secondary
axis
only
18
10 (56 %)
6(33%)
2(11%)
16
4(25%)
12 (75 %)
0
tentacles were removed in the usual way from both primary and secondary
axes, while in the second group these regions were removed from the secondary
axis only. The situation in the first group can be considered as equivalent to the
experimental procedure of transplanting suspected subhypostomal regions to
9
JEEM l6
130
G. WEBSTER
the digestive zones of hosts minus hypostome and tentacles; that in the second
group to performing a similar operation using intact hosts.
The secondary axis was so short at the time of cutting that after the wound
had healed the length of axis remaining was very much shorter than the piece
projecting in a usual transplantation experiment. Possible complications due to
axial distance effects could therefore be ruled out.
The number of animals possessing primary and/or secondary axes 48 h after
the operation is shown in Table 4.
It is clear that the subhypostomal region of the induced secondary axis
possesses the characteristic properties of this region, being absorbed in the
presence of the (primary) hypostome and forming a hypostome in its absence in
the majority of cases.
The suppression of primary hypostome formation in two cases and the
reconstitution of the secondary axis in the presence of the primary hypostome in
four cases may indicate that the new subhypostomal region has 'superior'
properties with regard to hypostome formation. However, it seems more likely
that these represent cases in which all the secondary hypostome was not
removed; the shortness of the axis made removal of the hypostome and tentacles
without any axis difficult, and in attempting to avoid removal of part of the
axis some of the hypostome may have been left in place in some animals.
Discussion of results of Experiment 4
The fact that an induced secondary axis possesses a region with typical
subhypostomal properties is direct evidence that regional properties are determined by the position of a region in relation to the hypostome. The proximity
of a hypostome has changed the time-threshold properties of part of the
digestive zone to those appropriate to the new axial position of the region. It
seems legitimate in this case to speak of the regional properties as being induced
by the hypostome.
Strictly speaking, the results only allow us to conclude that the time properties of the region have been changed, and that its T50 is now less than 9-5 h.
If, following hypostome removal, the level of inhibition in the digestive zone
falls below the threshold of the mid-digestive zone, then a new hypostome will
be formed by this region with only a change in T50 (see Exp. 1) and no change in
threshold. However, there is no evidence that the level of inhibition does fall
in this way, and taking into account the results of Exp. 2 which suggested that
time and threshold properties were closely linked, it seems more probable that
both properties have changed as a result of change in axial position.
Thus there would appear to be two ways in which the properties of a region
can change to those characteristic of more distal regions: (1) Spontaneously,
following isolation and preceding the formation of a hypostome (Exp. 2);
(2) by induction, following the acquisition of a new axial position in relation to
a transplanted hypostome.
Pattern regulation in hydra. Ill
131
Experiment 5. Changes in time-threshold properties of
subhypostomal region following transplantation
In exp. 1 of Webster (1966) it was demonstrated that a subhypostomal region
transplanted to the digestive zone of a host hydra formed a hypostome in the
absence of the host hypostome, but was absorbed in its presence. The purpose
of the present experiment was to study the time-course of the change in properties which accompany absorption.
Freshly isolated subhypostomal regions were transplanted to the digestive
zones of intact host hydra in the usual way. At 1,5- 5 and 9 h after transplantation
the hypostomes and tentacles of the host animals were removed.
The number of animals in which the graft produced a secondary axis is shown
in Table 5.
Table 5. Changes in time-threshold properties of a subhypostomal
region following transplantation to the digestive zone
Time after
grafting
when
hypostome
removed
(h)
1
5-5
9
No. of
successful
grafts
No. of
animals with
secondary
distal axes
20
20
20
16(80%)
6 (30 %)
1 (5 %)
Increasing the delay between transplantation and the removal of the host
hypostome and tentacles progressively reduces the frequency of hypostome
formation from the grafted subhypostomal regions. The approximate minimum
delay necessary for 50 % of the pieces to be absorbed can be estimated as 4 h.
Discussion of results of Experiment 5
This experiment provides confirmatory evidence that a change in the axial
position of a region is followed by a change in its time-threshold properties.
Transplantation of the sub-hypostomal region to a more proximal position
results in an eventual change in the Tb0 for this region from 4-5 h to more than
9-5 h, which is presumably accompanied by a corresponding change in the
threshold to that characteristic of the new axial position. This change in properties is associated with, or followed by, absorption.
9-2
132
G. WEBSTER
GENERAL DISCUSSION
The most important points to emerge from the above experiments are:
(1) Following the removal of the hypostome the level of inhibition falls
(Webster, 1966), and Exp. 1 above shows that hypostome formation is accompanied by restoration of the level of inhibition to that of the intact hydra. The
maximum time for this to occur is about 9 h. It may be as short as 5 h, which is
the time taken for a new hypostome to be determined in the subhypostomal
region. The time for restoration of inhibition must represent a maximum time
for hypostome determination and it is encouraging that this quite different
approach gives a similar time to that obtained previously.
(2) During the formation of a hypostome from the distal peduncle, the
threshold for inhibition rises in the presumptive hypostomal region. The region
appears to acquire the threshold properties of the subhypostomal region at the
time when it also has the same T50. This suggests that the factors controlling
time for hypostome formation and threshold are closely linked.
(3) During regulation it has not been possible to demonstrate changes in
threshold properties in regions other than the presumptive hypostomal region
until some time after the hypostome has formed.
(4) The time-threshold properties of a region are determined by its position on
the linear axis in relation to the hypostome.
We may now attempt a more detailed analysis of the processes involved in the
regulation of the hypostome in hydra.
In the intact hydra the hypostome is continually producing inhibitor and this
is continually lost or destroyed by the rest of the animal. This results in a
steady state with level of inhibition decreasing with distance from the hypostome.
The level of inhibition is well above the threshold throughout the animal.
The value of the threshold is determined by the position of a region on the
linear axis in relation to the hypostome. There seem to be at least two possible
mechanisms for this. One possibility is that the hypostome produces some
substance which by diffusion establishes a gradient which determines threshold.
The other possibility is that the hypostome polarizes the system. This polarity
determines the direction of movement of a substance (or substances) present
and transported actively by all the cells. This directional transport will also
establish a gradient (Webster & Wolpert, 1966&). It is of interest in this connexion that the hypostome or dominant region has an orienting influence. This
is clearly seen in the induction of a new axis and in budding (Webster, 1966).
It is possible that this orientation is intimately linked with the polarization of the
system. This suggestion is somewhat different to that of Rose (1957), who has
postulated polarized transport of inhibitory substances in Tubularia.
Following removal of the hypostome the level of inhibition falls. The details
of the changes in level throughout the system are not known. It is crucial to
determine whether, following hypostome removal, all regions are released from
Pattern regulation in hydra. HI
133
inhibition by the level of inhibition falling below threshold or whether only a
restricted region containing the presumptive hypostomal region is released.
In the former case, where all regions are released from inhibition, the polarity
of regulation would be determined by the time required for hypostome formation;
that is, the new hypostome would form at the distal end where the time for
hypostome determination is shortest. In the second case polarity is determined
by the threshold gradient. In this case the observed differences in time required
for hypostome determination could either reflect the time taken for hypostome
formation following release from inhibition (as in the previous case), or the
time taken for inhibition to fall below the threshold, or a compound of both.
In the absence of data on changes in level of inhibition throughout the
animal following hypostome removal, it is not possible to distinguish between
these alternatives. Moreover, there is as yet no criterion for determining whether
or not hypostome formation commences in regions other than the presumptive
hypostomal region. Changes in threshold during regulation may throw some
light on this problem.
In the case of regulation in the distal peduncle, it was shown that threshold
rises in the presumptive hypostomal region prior to hypostome determination.
The observation is not easy to interpret since it is not known when this occurs
in relation to the time when the region is released from inhibition. One possibility is that increase in threshold is one aspect of hypostome formation.
Following release from inhibition, threshold would rise, and the gradient in
threshold could be the factor responsible for the difference in time for hypostome determination, since proximal regions have lower thresholds than distal
regions and might be expected to take a longer time to obtain a higher threshold
value. The failure to obtain any evidence for threshold rise in regions other than
a presumptive hypostomal region may indicate that they were not released
from inhibition.
The observation that threshold rises during hypostome formation could by
itself account for those transplantation experiments in which the time for
hypostome determination was measured (Webster & Wolpert, 1966a). Once
the threshold had risen sufficiently the region would resist absorption since it
seems that a piece is absorbed only when its threshold is below the level of
inhibition. However, the fact that inhibition is restored at the same time as
hypostome determination, or within a short time after it, means that the new
hypostome produces inhibitor and suggests that a qualitative change in properties must have occurred in addition to an increase in threshold.
It has been assumed (Webster, 1966) in the development of the concept of
threshold that the threshold of a piece is more stable than the level of inhibition;
when a piece is transplanted its level of inhibition changes rapidly to that of the
site of transplantation while the threshold tends to persist for a longer time.
There is a no direct evidence for this assumption. Some support comes from the
observation that the maximum time for the fall in inhibition in a subhypostomal
134
G. WEBSTER
region following removal of the hypostome is about 4 h (Webster & Wolpert,
1966a), while the minimum time for a fall in threshold in a subhypostomal
region transplanted to the mid-digestive zone is 4 h.
The concepts for control of hypostome formation outlined above can be used
to explain many other cases of regulation following grafting. The literature on
hydra abounds with such experiments, but in this section only two will be
considered, both involving reversal of polarity.
(a) Reversal of polarity
Many workers on hydra have observed that polarity can be reversed by
grafting, and that the new polarity is determined in relation to the position of a
hypostome (Peebles, 1900; King, 1901; Browne, 1909; Goetsch, 1929; Tardent,
Fig. 1. Reversal of polarity. Left, a small piece grafted with reversed orientation
remains inhibited. Right, the distal end of a larger piece is released from inhibition
and a new hypostome is formed from the shaded region.
1954); this was also observed in exp. 2 of Webster (1966). A common experimental procedure has been to remove a part of the axis and replace it with
reversed orientation. It has often been remarked that this procedure will only
result in a stable and lasting combination if the piece which is reversed is of
small size. Reversal of a large piece produces an unstable system which eventually breaks up into three separate individuals.
The effect of size on stability is easily explained in terms of gradients in
inhibition and threshold (Fig. 1). When a small piece is reversed, the level of
inhibition, which is a function of distance from the hypostome, will remain
above the maximum (distal) threshold of the piece and it will remain inhibited.
This situation may be compared with that in exp. 1 of Webster (1966), where a
subhypostomal region grafted to the digestive zone remained inhibited as long
as the host hypostome was present. Exactly as in this situation, the time-
Pattern regulation in hydra. Ill
135
threshold properties of the various levels of the reversed piece will change to
conform to their new axial positions, those of the former distal end falling, and
those of the former proximal end rising. This will lead to a true reversal of
polarity, and, if the piece is eventually removed, it will regulate with a new
polarity. Davis (1963) has shown this to be the case. A stage will occur during
reversal of polarity when distal and proximal time-threshold properties are
equivalent, and it can be predicted that if such a piece is removed after an
appropriate time-interval bipolar regulation will occur. In the case of reversal
of a large piece, however, the situation will be different. The distal end of a large
piece will be removed to such a distance from the hypostome that the level of
inhibition will be below the threshold and the region will be released from
inhibition. A hypostome will form and organize part of the 'host' axis in relation to itself to produce a new individual which will separate by some unknown
mechanism. The separated distal and proximal parts of the host will also undergo
regulation to restore the missing parts, and three individuals will be produced.
(/?) King's experiment
King (1901) performed several experiments in which polarity was reversed.
One of the most interesting involved grafting together two hydra (from which
the peduncles and basal discs had been removed) with opposite polarity, i.e. by
the proximal cut ends of the digestive zones. The usual result in such an experiment was that new basal discs were produced at the junction and the two animals
eventually separated. If, before separation occurred, the hypostome and
tentacles were removed from the right-hand component by a cut made just
below the ring of tentacles, regeneration of the missing regions occurred in the
usual way. However, if the cut was made nearer the junction between the two
individuals, the remaining portion of the right-hand component did not produce hypostome and tentacles but transformed into a peduncle and basal disc.
These results can be explained (Fig. 2) if we assume that the gradients in
threshold in the two components remain unchanged (Tr and 7]), but that the
gradient in level of inhibition in each component is extended into the opposite
member of the pair (1^ and Ii). When the hypostome of the right-hand component is removed by cut A (made just proximal to the tentacles) the level of
inhibition due to this component will fall below all thresholds to Irl. The distal
region of the right-hand component is at such a distance from the hypostome
of the left-hand component that the level of inhibition (/,) due to this component will also be below its threshold. The distal region will therefore be released from inhibition and form a hypostome in the usual way. However, when
the cut is made nearer the junction (cut B), the course of events will be exactly
as described above, but in this case the most distal region of the right-hand
component is at such a distance from the left-hand hypostome that the level
of inhibition (/,) will be above its threshold and it will be prevented from forming
a hypostome. In the absence of its own hypostome, the remaining portion of the
136
G. WEBSTER
right-hand component will come under the control of the left-hand hypostome,
and reversal of polarity will occur (changes in time-threshold properties) as
described above. The region will form the structures appropriate to its proximal
position: peduncle and basal disc.
B
I
n
I
u
A
Fig. 2. King's experiment (see text).
Relation to work on other hydroids
Watanabe's (1935) data on the reconstitution of hydranths in Corymorpha
enable an estimate of the time for restoration of the level of inhibition to be
made. For a hydranth reconstituting at a distal end, the T50 for inhibition of a
proximal region is about 12 h. For a hydranth developing at a proximal end
the r 50 for inhibition of a distal region is about 24 h. These times are of the same
order of magnitude as those found in hydra. Corymorpha also resembles hydra
in several other aspects of reconstitution (Webster & Wolpert, 1966c; Webster,
1966).
Comparison with Tardent & Eymann's (1959) work on Tubularia is difficult
since they were concerned with effects on rate of total reconstitution rather than
* all-or-none' inhibition. They investigated the restoration of inhibition by
recording the degree of inhibitory interaction between regenerates at different
developmental stages which had been grafted together. Using rate of regeneration
Pattern regulation in hydra. Ill
137
as a criterion they showed that inhibition increases as regeneration proceeds
and reaches a maximum level just before visible differentiation (tentacle ridges)
appears.
With regard to changes in threshold some other results of Tardent & Eymann
(1959) on Tubularia are of interest. In graft combinations of regenerates at
different stages of development, the resistance to inhibition, judged by the
effect on the rate of regeneration, by a given stage (stage E, proximal tentacle
ridges) progressively increased during regeneration until at a certain stage complete resistance was acquired and the experimental animals regenerated at the
same rate as the controls. From Tardent & Eymann's diagram, complete
resistance seems to be acquired at or about the time when the distal primordium
is determined (Steinberg, 1954). Although these workers used rate of total
reconstitution as a criterion of the effect of inhibition, thus making their
observations somewhat difficult to compare with the present ones on hydra,
their findings are strongly suggestive of an increase in threshold during regeneration, culminating in ' absolute resistance' to inhibition just after the formation
of the distal organizer.
A model for proportionate regulation in a two-compartment system
The major aim in this series of papers was to develop concepts to explain the
regulation of a two-compartment linear pattern. The study of the factors
regulating hydra into hypostomal and non-hypostomal regions has shown that
the two main factors involved are gradients in inhibition and threshold. While
no data has been obtained that is specifically relevant to the problem of proportionality, the general impression in all the experiments was that proporportionality was restored. It is thus relevant to consider how one can derive a
mechanism for controlling proportionality using the concepts developed.
Consider a system consisting of a linear array of cells which is divided into
two compartments, A and B. Transformation between types A and B is reversible and is determined by the following conditions. A cells produce a substance
/ and B cells destroy /. Whether a cell shall be A or B is determined by the level
of / ; if / is above a threshold then B cells cannot become A; if / is below the
threshold then they transform into A. The threshold varies continuously and
decreases monotonically from one end of the system to the other. / is freely
diffusible within the system. Type A cells produce / at a fixed rate per cell kA;
type B cells destroy or metabolize / and remove it from the system also at a
fixed rate per cell kB. For simplicity we will assume that transformation of cells
to type B is accompanied by cessation of / production, and that the rate of
transformation, A -> B or B -» A is the same for all cells.
In a steady state, when transformation has ceased, the equilibrium conditions
will be as follows. The overall rate of production of/will be equal to the overall
rate of destruction, i.Q.nAkA = nBkB, when nA and nB are the number of A and
B cells. It is clear that nA\nB = kB\kA i.e. the proportions of A to B are deter-
G. WEBSTER
138
mined by the ratio of the rate constants for production and destruction of /
per cell.
Such a system will show polarized regulation (Fig. 3). If the distal and
proximal halves are isolated, each will reorganize to form a complete system of
normal proportions. In the isolated distal half, there are no type B cells and
therefore /is not destroyed. The concentration rises and transformation to type
B occurs when the threshold is exceeded. It is clear that transformation will
(fa)
Fig. 3. Proportionate regulation in a two-compartment system, (a) The intact
system: all parts of region B (unshaded) are prevented from transforming into A
(shaded) since the level of/is above the threshold T. {b) Region A has been removed
and / has fallen below the threshold at the left hand side of region B, thereby permitting part of B to transform into A (shaded), (c) A new equilibrium has been
established and transformation of B into A has ceased: proportionality has been
re-established at a lower level of /. (d) Threshold has risen and has been accompanied by an increase in the level of / to that of the original system.
proceed in the direction of increasing threshold, i.e. in a proximo-distal direction, and therefore the new B region will be formed at the proximal end of the
system. In the isolated proximal half no / is being produced, so that the concentration falls. Cells will transform back to type A when the concentration of/
Pattern regulation in hydra. Ill
139
is below the threshold, and the direction of transformation will be distoproximal, the direction of decreasing threshold, so that the new A region will
form at the distal end of the system. Once again transformation will cease at
equilibrium when the concentration of / ceases to fall.
Thus in a system in which the only initial difference between the cells is in
threshold for response to /, we have obtained polarized and proportionate
regulation into two spatially distinct compartments. It will be apparent that the
precise values of the thresholds, i.e. the slope of the gradient, are immaterial
since the role of thresholds is merely to provide differences. They play no active
role in transformation, but simply ensure that it occurs in only part of the
population, and ceases when / has reached a constant equilibrium concentration. The threshold gradient determines the polarity of the regulation. Not only
is the slope of the gradient unimportant, but its precise shape does not have to
be carefully controlled; the gradient is drawn as linear in Fig. 3 but this is
purely for simplicity. There is no reason why the slope and the shape of the
gradient should not differ markedly from one system to another, but the
systems will always show the same proportionality.
The regulation in the isolated proximal region can be considered analogous
to hypostome regulation in hydra. In the model as proposed, regulation results
from a fall in level of the inhibitor / to a new equilibrium position. In hydra it
was shown that the initial fall in inhibitor level is followed by a restoration to
the original level. This could only occur in the model if the threshold gradient
was restored. This is consistent with the observation that threshold rises during
regulation in hydra.
Further aspects of the model will be considered elsewhere (Webster &
Wolpert, 19666).
SUMMARY
1. The dynamic behaviour of the factors (level of inhibition and threshold
for inhibition) controlling hypostome formation in hydra has been investigated during regulation using transplantation techniques.
2. The formation of a new hypostome following removal of the original
hypostome is accompanied by restoration of the level of inhibition to that of the
intact hydra. The maximum time for them to occur is about 9 h. It may be less—
about 5 h which is the time taken for a new hypostome to form from the subhypostomal region, as shown in a previous paper.
3. During the formation of a hypostome from the distal peduncle the threshold for inhibition rises in the presumptive hypostomal region. The results
suggest that the factors controlling the time for hypostome formation and
threshold for inhibition are closely linked.
4. It has not been possible to demonstrate changes in threshold properties in
regions other than the presumptive hypostomal region until some time after the
new hypostome has formed.
140
G. WEBSTER
5. The time-threshold properties of a region are determined by its position
on the linear axis in relation to the hypostome.
6. The significance of the results in relation to polarized regulation in hydra
and other hydroids is discussed, and the concepts developed are used to explain
some cases of reversal of polarity following grafting.
7. A model for proportionate regulation in a two-compartment system is
presented, based on the concepts of control developed for hydra.
RESUME
Etudes sur la regulation chez l'hydra. III. Aspects dynamiques des
facteurs controlant la formation de Vhypostome
1. Le comportement dynamique des facteurs (force de l'inhibition et seuil de
l'inhibition) controlant la formation de l'hypostome chez l'hydra a ete etudie
pendant la regulation au moyen de technique de transplantation.
2. La formation d'un nouvel hypostome apres ablation de l'hypostome
primitif est accompagnee de la restauration de la force inhibitrice, comme chez
l'hydre intacte. Le temps maximum necessaire a cette restauration est d'environ
9 heures. II peut-etre inferieur — environ 5 heures — ce qui est le temps requis
pour qu'un nouvel hypostome se forme a partir de la region sous-hypostomale,
comme cela a ete montre dans un precedent article.
3. Pendant la formation d'un hypostome a partir du pedoncule distal le
seuil d'inhibition s'eleve dans la region hypostomale presomptive. Les resultats
suggerent que les facteurs controlant le temps necessaire a la formation de
l'hypostome et le seuil de l'inhibition sont tres etroitement lies.
4. II n'a pas ete possible de demontrer des modifications dans les proprietes
du seuil dans des regions autres que celle de l'hypostome presomptif tant qu'un
certain laps de temps ne s'est pas ecoule apres la formation du nouvel hypostome.
5. Les proprietes temps-seuil d'une region sont determinees par sa localisation sur l'axe lineaire par rapport a l'hypostome.
6. La signification de ces resultats relatifs a une regulation polarisee chez
l'hydre et chez d'autres hydroides est discutee, et les concepts developpes sont
utilises pour expliquer quelques cas de renversement de la polarite apres greffe.
7. Un type de regulation proportionnee en un systeme a deux compartiments est presente, sur la base des concepts de controle developpes pour l'hydre.
I should like to thank Dr Lewis Wolpert for his advice and encouragement during the
course of this work and the Agricultural Research Council for a Postgraduate Research
Studentship.
Pattern regulation in hydra. Ill
141
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(Manuscript received 28 January 1966)