/. Embryol. exp. Morph. Vol. 33, 2, pp. 511-521, 1975
Printed in Great Britain
Positional information and
pattern formation in Hydra. Dynamics of regions
away from the boundary
By J. HICKLIN, 1 A. HORNBRUCH 1 AND L. WOLPERT 1
From the Department of Biology as Applied to Medicine,
The Middlesex Hospital Medical School
SUMMARY
The dynamics of regions away from the head-end boundary have been investigated by a
variety of assays, and the changes appear to be slow. If, for example, a head end is grafted
onto a peduncle to give H/56F, region 5 does not become a region 1 within 72 h. Such
results support a gradient type model, and exclude a head end inducing a region 1 in the
tissue adjacent to it. The changes can be interpreted if the positional value were effectively
slowly diffusible: about 10 times more slowly than the positional signal. These results also
have implications for studies on polarity reversal.
INTRODUCTION
On the basis of grafting experiments (Webster, 1971; Wolpert, Hicklin &
Hornbruch, 1971; Hicklin, Hornbruch, Wolpert & Clarke, 1973) a two-gradient
model has been proposed for the regulation and regeneration of hydra (Wolpert,
Hornbruch & Clarke, 1974). One gradient is assumed to be of a diffusible
morphogen made at the head end, which may be regarded as a positional signal
S, and the other is a more stable cellular parameter which is the positional
value, P. Much of the work has been directed towards head-end regeneration
and the rule for initiating head-end formation is that S should fall a threshold
amount below P. This can account for head-end formation in a variety of axial
grafts (Hicklin et al. 1973). In a previous paper (Hicklin, Hornbruch & Wolpert,
1975) we investigated the changes with time that occurred at a regenerating head
end. These were the ability to resist inhibition of head-end formation and the
acquisition of inhibitory properties. The results were, in general, similar to those
obtained by lateral grafting (Webster, 1966 a, b; Webster & Wolpert, 1966), but
there was evidence that resistance to inhibition occurred prior to the ability to
inhibit. For regions in the gastric zone the times required to escape from inhibition were of the order of 10 h. In terms of the model, the head end is only
1
Authors' address: Department of Biology as Applied to Medicine, The Middlesex
Hospital Medical School, London, W1P 6DB, U.K.
512
J. HICKLIN AND OTHERS
fully determined when P has increased to a value corresponding to that of the
head-end boundary. Before that time, head-end formation can be prevented if S
is increased sufficiently: S effectively acts as an inhibitor of head-end formation.
Acquisition of inhibitory properties involves synthesis of S at the head end, and
this occurs mainly when the head end is fully determined.
An important question is how localized are the changes that occur during
head-end regeneration. Do they occur only at or near the regenerating end or do
they occur throughout the hydra? In terms of the model this involves asking
questions about the changes in P away from the boundary region. For example,
while region 1 (the hydra is represented as H1234B56F as in previous papers) is
increasing to become an H, what is happening in region 2 adjacent to it? Does
region 2 simultaneously increase its P to become region 1 ? Another important
question is the effect of a head region when grafted onto regions with which it is
not normally in contact. For example, what happens to the 3 region of an
H/3...F combination? This experiment has also implications for the experiments of Wilby and Webster (1970) in which it was found that the reversal of
polarity of the 123 region in an H/321 graft occurred only very slowly. For
polarity reversal, it is likely that the 3 region has to increase its positional value
under the influence of the H. The necessity to understand this interaction was an
important stimulus for the experiments described in this paper.
In order to determine the changes with time of a region, such as region 3,
away from the boundary, the standard assay of time for head-end determination
cannot be used. We do have, however, an assay for region 1 and can thus
determine the time for the 3 region to become a region 1. The assay for region 1
is based on Webster's (1966 a, b) studies which showed that if region 1 were
grafted into the gastric region of a hydra from which the head had been removed, it would induce a secondary axis. In the presence of a head it was, of
course, absorbed.
MATERIALS AND METHODS
All experiments were performed using Hydra littoralis as described in Hicklin
et al. (1973) and Hicklin & Wolpert (1973) and all incubations were at 26 °C.
The results are scored in terms of structures at the junctions between grafts and
those formed following lateral grafting. Very briefly, N signifies normal development with no structures at the junction: H is head, HF is head and foot, F is
foot at the junction, R refers to formation of a foot at the distal end and I to
the absence of any distal structures.
RESULTS
Behaviour of region 1 away from the boundary
Grafts of HI2/12...F do not form structures at the junction, whereas head and
foot ends form at the junction in a high proportion of 12/12...F (Hicklin et al.
Dynamics of regions away from boundary in Hydra
513
Table 1. Grafts of HI2/12...F followed by removal of H
Time before
H removed
(h)
No. of
combinations
N*
H
HF
F
4
6
8
10
12
15
16
23
15
24
6(40%)
9(56%)
22(96%)
15(100%)
24(100%)
7(46%)
2(13%)
1(4%)
—
—
1 (7 %)
5 (31 %)
1 (7 %)
-
Results
* A significant number of the heads regenerated at the distal end were abnormal.
Table 2. H onto regenerating 12J12...F
Time after
which H grafted
No. of
on 12/12...F
combinations
(h)
1
3
5
7
9
20
19
18
16
12
Results
N
H
20 (100 %)
15(79%)
15(83%)
9 (56 %)
3 (25 %)
0
3(16%)
3(17%)
7 (44 %)
8 (67 %)
F
0
1(5%)
0
0
1 (8 %)
FH
R
0
0
0
0
0
0
0
0
0
0
1973). One can then ask how long the graft combination H12/12...F must be
left before removal of the H will no longer lead to the formation of structures at
the junction. Grafts of H12/12...F were prepared and at various times afterwards
the H was removed. The results are given in Table 1. When the head was
removed 6-8 h after grafting, there was a marked change in the proportion of
animals forming structures at the junction between graft and host. Whereas nine
out of fifteen animals formed structures at the junction where the head had been
removed after 4 h, only one animal out of the twenty-three belonging to the 8 h
series gave this result. However, it was observed that many animals did not
regenerate a head of normal appearance at the distal end of the graft. Instead of
forming a neat ring about a terminal hypostome, tentacles often regenerated on
a disorganized pattern scattered from the tip to about halfway down the region 1
of the graft. In such animals, the mouth was often either absent or had regenerated some distance from the tip. Sometimes two or more tentacles appeared to be
fused together. While recognizing that wound healing at the junction may play a
role, this experiment suggests that about 6 h may be required for the P value of
the proximal region 1 to fall to about that of region 3. The effect of the host on
distal regeneration of the graft is not easily explained, but it may be due to such
factors as the abnormal distribution of S during regeneration.
It is of interest to consider how long it takes for the proximal region 12 in a
514
J. HICKLIN AND OTHERS
Table 3. Grafts of Hj3112...F following removal of H
Time before
H removed
(h)
No. of
combinations
Results
N
0
12
24
48
72
10
13
15
15
13
2 (20 %)
2 05%)
7 (46 %)
15 (100%)
13 (100 %)
R
4 (40 %)
1 (8 %)*
6 (40 %)•
—
—
F
I
1 (10%)
5 (39 %)
2 (14 %)
—
—
3 (30 %)
5 (38 %)
1 (7 %)
—
—
* No head regenerated at junction.
Note that I and F contain a few animals in common.
12/12...F combination to become determined as a head end with respect to the
presence of an H region at the end. To find out, 12/12...F combinations were
prepared and at various times an H region was grafted on the distal end. The
results in Table 2 suggest that about 7 h are required. The changes in a diffusible
S in such an experiment are complex and will require careful modelling.
Another way of looking at the behaviour of region 1 away from the head end
is in H/3/12...F combinations. These do not usually form structures at the
junction. By contrast, 3/12...F very often do so and in some cases the polarity of
the 3 region becomes reversed (Hicklin et al. 1973). We therefore examined the
time course of this change in behaviour by preparing H/3/12...F combinations
and removing the H at various times. The results are given in Table 3 and show
that the presence of the head was necessary for about 24 h before there is a
significant increase in the number of normal animals. After 12 h no heads form
at the junction. The relative contribution of the P of region 1 falling, or region 3
rising is not known, but there are a significant number of polarity reversals and
presumably some averaging is occurring.
Dynamics of region 3 away from the boundary
As pointed out in the Introduction, the changes with time of a 3 region can be
assayed by determining the time it takes to become a 1 region. The assay for
region 1 is that lateral grafts of this region will induce distal axes in the gastric
regions of headless hosts (Webster, 1966a). The time for a region 3 to become
a region 1 was first determined for region 3 at the boundary. HI2 was removed
and at various times the 3 region was grafted into the gastric region of headless
hosts. The results are given in Table 4 and all positive inductions are distal. It
can be seen that the results are rather variable but that it takes about 14 h for
region 3 to become region 1 by this assay. This time was rather longer than
might be expected since, with other batches of hydra, the time for head-end
determination, using lateral grafting was 11-12 h (Hicklin et al. 1975). It should
Dynamics of regions away from boundary in Hydra
515
Table 4. Determination of 1 from region 3 of regenerating 34...F
Regeneration
(h)
No. of grafts
Absorbed
Distal inductions
0
4
8
10
12
14
16
18
20
10
10
10
10
10
10
10
10
10
10
9
10
3
4
5
4
3
—
1
—
7
6
5
6
7
10
Note that a significant number of animals do not regenerate heads at their distal end.
Table 5. Determination of 1 from region 3 of HJ34...F
Time before
Inductions
(h)
No. of grafts
Absorbed
H
F
18
24
48
72
96
24*
48*
72*
96*
10
20
20
10
10
10
10
11
10
10
19
19
10
10
10
10
9
8
—
—
—
—
—
—
—
2
2
—
1
1
—
—
—
—
—
—
* Giafts were fed daily.
also be noted that distal regeneration was again sometimes inhibited by the
induced axis.
In order to determine the time for the 3 region to become region 1 when it is
away from the boundary, two situations were used: H/34...F and regenerating
23...F. Combinations comprising H/34...F were prepared, and at successive
times the 3 region was assayed by grafting it into the gastric region of headless
animals. The results are shown in Table 5. The surprising result is that there are
virtually no positive inductions even after 3 days. The same was true even when
the graft combinations were fed. This result was briefly reported in Wolpert et ah
(1971) and has been confirmed by Wilby & Webster (Webster, 1971). In order
to make sure that the absence of a free cut surface was not playing a major role,
the time for region 3 to become region 1 in F...43/34...F combinations was
measured (Table 6) and it can be seen that by 15 h most grafts induced distal
axes.
516
J. HICKLIN AND OTHERS
Table 6. Determination of 1 from 3 of F...43/34...F
Regeneration
(h)
No. of grafts
Absorbed
10
12
15
21
24
4
6
10
10
10
4
4
2
2
1
Induced H
—
2
8
8
9
Table 7. Determination of 1 from 3 of a regeneration 23...F
Regeneration
time
Inductions
00
No. of grafts
Absorbed
18
10
10
25
30
42
10
10
10
9
9
9
( H
—
—
1
1
F
—
1
—
—
Table 8. ,Determination of 1 from regenerating 56...F
Inductions
Regeneration
00
No. of grafts
Absorbed
H
F
6
16
20
24
28
10
10
10
10
10
—
—
2
1
—
—
1
6
8
10
10
9
2
1
—
The very long time required for a region 3 to become a region 1 when it is
adjacent to the boundary region was confirmed by experiments using regenerating 23...F. At various times the 3 region was assayed for region 1 properties
(Table 7). It was found that even although tentacles had regenerated at the
distal end of the animal the region just beneath the head, the original 3 region,
did not behave as a region 1 after extended periods of regeneration.
Dynamics of region 5 away from the boundary
Experiments similar to those just described for region 3 were carried out on
region 5. First, the time required for region 5 to become like a region 1 was
determined using the lateral grafting assay (Table 8). About 16-20 h were
required and this is slightly longer than that reported in earlier experiments
(Hicklin & Wolpert, 1973), in which region 5 could induce distal axes in intact
animals after about 16 h.
Dynamics of regions away from boundary in Hydra
517
Table 9. Determination of 1 from region 5 of Hj'56F
Inductions
Time before
(h)
No. of grafts
Absorbed
24
48
72
96
144
10
12
11
10
9
3
8
11
10
H
1
9
B
H
—
2
—
—
—
F
7
2
—
—
—
5 6
100
80
60
40
20
100
80
60
40
20
Fig. 1. Diagram to show the possible changes in a gradient in positional value in
H/34...F and H/56F in relation to formation of a new region 1. (A) shows the
postulated gradient in the intact hydra. If a linear gradient is established in H/34...F
(B) and in H/56F (C) the region with an average value equivalent to a region 1 is
either at the graft boundary in (B) or within the original H region (C).
The time required for region 5 to become like region 1 when it is in a H/56F
combination is shown in Table 9, from which it can be seen that even after a very
long time, 144 h, region 5 does not induce distal axes when grafted into headless
hosts.
DISCUSSION
The most significant result obtained was that increases in positional value
away from the boundary appear to occur very slowly. Placing a head end
adjacent to a region further down the hydra, such as region 3 or region 5, does
518
J. HICKLIN AND OTHERS
100
90
80
70
60
100
90
80
70
60
12
16
20
24
28
Time (h)
Fig. 2. Changes in the value of P by diffusion following grafting. A computersimulated model of a 17-cell hydra was used as in Hicklin et al. (1973). The diffusion
constant is taken at 2 x 10~8 cm2/sec. (A) Changes at the proximal end of the H (A)
and the distal end of region 3 (A) in H/34...F. (B) Changes at the distal end of region
1 (O) and the distal end of region 3 ( # ) in H/3/12...F. (We are indebted to M. R. B.
Clarke, Department of Computer Science, Queen Mary College, University of
London, for these computations.)
not result in a rapid increase in the positional value of that region. This at once
excludes mechanisms in which a head region can rapidly induce a region 1. The
results are better looked at in terms of a mechanism based upon gradients
which provide positional information. If we assume a linear gradient, then in an
H/34...F graft it can be seen that if gradient is restored (Fig. 1) the new region 1,
as defined by its P value, will be from both the 34...F and the H of the initial graft
combination. It is thus perhaps not surprising that region 3 gave so few indications that it had become a region 1. This is even more true of the H/56F grafts in
which, on the gradient model, region 1 would come from the H. Since region 5
can become like a region 1 in 20 h when H is absent, it is clear that the presence
of the H not only does not induce region 1 in the region adjacent to it but
Dynamics of regions away from boundary in Hydra
519
prevents region 5 from becoming region 1. This would seem to constitute quite
good support for considering hydra in terms of gradients.
The regeneration of 23...F provides more direct evidence for the localized
increase in P and the very slow rise in P in the region adjacent to the new H.
The slow change in region 3 when adjacent to an H is also seen in H/3/12...F
combinations: here the gradient is more spread out and it takes about 24 h for
region 3 to have increased sufficiently for normal regeneration to occur after
removal of the H.
There does, however, seem to be some evidence for a more rapid decrease in
P away from the boundary as shown by the behaviour of H12/12...F after
removal of the H. There the changes occur in about 6 h.
In all our considerations we have treated P as a more stable parameter than S,
and while recognizing that it may be diffusible (Wolpert et al. 1971) have usually
made the simplifying assumption that it does not diffuse, since the experimental
results did not require it. In considering how P changes away from the boundary, the possibility that P is slowly diffusible must now be considered. The
other possibility is that changes in P occur by synthesis and destruction and
involve a follow-up servo mechanism, P being specified by S (Wolpert, 1971).
We cannot decide between these mechanisms but a slowly diffusible P seems a
simpler mechanism. Consider, for example, the diffusion of P in H/34...F and
H/3/12...F if P has a diffusion coefficient of 2 x 10~~8, that is 10 times slower than
that assumed for S. Fig. 2 A shows the changes at either side of the junction in
an H/34...F, that is the proximal end of the H region and the distal end of the
3 region. After 10 h region 3 has only reached a level of 75 which is significantly
below that of a region 1. In the H/3/12...F, the changes of the distal end of the
3 and 1 regions are given in Fig. 2B. It is only after about 20 h that the P value
of the 3 region is significantly above that of the 1 region and this is in accord
with the observations following removal of H. In H12/12...F combinations the
distal end of the proximal 1 falls by about 10 % below the distal region 1 in 10 h
(see fig. 8, Hicklin et al. 1973) and this could account for the behaviour of such
combinations following the removal of the H. Slow diffusion of P could also
help to explain the induction of a new axis by lateral grafting of a head end.
The value of the diffusion constant for P chosen here is obviously only an
approximation and the times are directly proportional to it. It should also be
remembered that we are dealing with the effective diffusion of the P value and
this could involve quite complex interactions of numerous chemical species
between adjacent cells.
Our results may have important implications for the mechanism responsible
for reversal of polarity, particularly in relation to the experiments of Wilby &
Webster (1970). They found that although a head grafted to the proximal end of
a gastric region to give H/321 could prevent head-end formation from region 1,
true polarity reversal of the region 321 following removal of the grafted H
required about 72 h. They also found that in H/321/56F combinations polarity
520
J. HICKLIN AND OTHERS
reversal was faster. An important observation, following removal of the grafted
H, was the formation on occasion of a medial hypostome with a foot regenerated
at both ends. From their results they concluded that any model based on a
diffusion mechanism could not account for the results and argued for a model
based upon active transport. As we have pointed out before (Wolpert et al.
1971), we consider that they have not fully explored the implications of a two
gradient model in which one gradient (S) is of a relatively rapidly diffusing
morphogen and the other (P) changes primarily by synthesis and destruction,
and diffuses much more slowly. We consider polarity, as assayed by observing
which end of an isolated fragment forms a head end, to be dependent on the
interaction between the two gradients, since in our model head-end formation
is initiated when S falls a threshold amount below P. While not claiming to be
able to account for the polarity reversal results of Wilby & Webster, some
features at least can be accounted for and are consistent with our model.
When a head is grafted to the proximal end of a gastric region as in H123/H,
the time between effecting the head graft and removing the host's head, so that
an inhibitory signal from the grafted head can inhibit head-end formation, is
consistent with diffusion of S from the grafted head (Wolpert, Clarke & Hornbruch, 1972). A plausible estimate of the time required for S to be distributed
along such a hydra to achieve a concentration high enough to inhibit head-end
formation is about 10 h. We cannot comment on the distribution of S at later
times since there is no sink present and it seems easiest to assume a uniform high
concentration. For true polarity reversal to occur with this distribution of S,
there must be a reversal of the P gradient: the positional value of P at 1 must go
down and that of 3 must increase. As regards the increase in 3, the results we
have presented here suggest that it is very slow and this may play a major role in
determining the time taken for polarity reversal. There is no reason to believe
that the behaviour of region 3 adjacent to a head end is in any way dependent
on its polarity in relation to that head. The fall in P at the 1 region may, like the
rise in the 3 region, be due to diffusion, but we would anticipate that it would be
slow. For reversal of polarity this region must become a foot end, and other
considerations apart, we have shown (Hicklin & Wolpert, 1973) that region 1
of a HI takes both a very long time to become determined as a foot end and is
very poor at regenerating a foot end at all. We do not know how this determination of the new foot end, as well as the behaviour of incipient buds, affects the
behaviour of the system under these unusual conditions. What is clear is that
at this stage, it is unnecessary to invoke mechanisms such as active transport to
account for polarity: mechanisms based on the interaction of two diffusion
gradients will probably suffice. In this connexion it should be remembered that
reversal of polarity can occur even with pieces grafted with the same polarity:
56/12...F gives feet at both ends and 123/1 gives heads at both ends (Hicklin
et al. 1973).
Dynamics of regions away from boundary in Hydra
521
This work is supported by the Nuffield Foundation.
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