/ . Embryol. exp. Morph. Vol. 33, 2, pp. 499-510,1975
Printed in Great Britain
499
Positional information and
pattern regulation in Hydra. Dynamics of regions
at the boundary
By J. HICKLIN, 1 A. HORNBRUCH 1 AND L. WOLPERT 1
From Department of Biology as Applied to Medicine,
The Middlesex Hospital Medical School
SUMMARY
The dynamics of boundary regions have been investigated mainly by axial grafting and
with the emphasis on the head end. The time to lesist inhibition of head-end formation and
the ability to inhibit head-end formation have been assayed under a variety of conditions.
The times increase with distance from the head end. The times required by a boundary region
to acquire the inhibitory properties of a head end are longer than those required to acquire
resistance to inhibition. Determination of a head end is faster at a cut surface and at higher
temperatures. The results are discussed in terms of a model involving two gradients. Some
anomalous results are reported.
INTRODUCTION
In a previous paper (Hicklin, Hornbruch, Wolpert & Clarke, 1973) we considered the results of axial grafts of hydra in terms of a model based on two
gradients, one in a diffusible positional signal S, and the other in a more stable
parameter, the positional value P. It was suggested that the results could be
interpreted on the basis that a new head end would form if S fell a threshold
amount below P. One can think of P as a parameter which tells a cell what state
it currently is in, and S as a parameter which can respond to the state of neighbouring cells (Wolpert, Hornbruch & Clarke, 1974). In the model, S is assumed
to be synthesized at the head-end boundary and it effectively acts as an inhibitor
of head-end formation elsewhere. Evidence has recently been obtained that the
transmission of this inhibitory influence is consistent with a mechanism based on
diffusion (Wolpert, Clarke & Hornbruch, 1972).
In order to construct a model it is necessary to know how P and S change
with time. These cannot, at present, be investigated directly and their dynamics
can only be inferred from experiments providing information on the change in
properties of regions with time. In this paper, we investigate the dynamics of the
1
Authors' address: Department of Biology as Applied to Medicine, The Middlesex
Hospital Medical School, London, W1P 6DB, U.K.
32
EMB 33
500
J. HICKLIN AND OTHERS
100
80
60
40
20
0
(a)
(b)
(c)
(</)
(e)
Fig. 1. Diagram to illustrate the assumed relation between S and P during regeneration. S(a) shows the equilibrium condition. Following the removal of the distal
region the concentration of S falls until it reaches a threshold amount below P(6).
This initiates synthesis of P and also a small amount of S. P increases now to the
boundary value, but if S rises before this value is attained, synthesis of P is inhibited.
Thus, in (c) an increase of S could block further synthesis of P. Once P reaches the
boundary value as in (d) S is synthesized at the boundary and increases until a linear
gradient is re-established (e). Note that no mechanism is proposed here for changes
in P away from the boundary.
boundary region. The boundary region is that region which will become a head
end or a foot end. The properties, whose change with time we assay, are
resistance to inhibition and the ability to inhibit. Preliminary results were
briefly reported in Wolpert, Hicklin & Hornbruch (1971).
The basic approach is that adopted in earlier papers (Webster & Wolpert,
1966; Webster, 1966a, b; reviewed Webster, 1971), and involves determining
how long a region takes to become determined as a head end (in the sense that it
can no longer be prevented from forming a head end in the particular graft
combination used), and how long a region takes to acquire the ability to inhibit
head-end formation elsewhere. These parameters can be considered in terms of
our current model (Wolpert etal. 1974), which is partly based on the results to
be presented here, though it must of course be remembered that other models
are possible. The model assumes that the 'switch' associated with resistance to
inhibition only occurs when P has increased to the value of the head-end
boundary. It is assumed that at any time prior to this, a sufficient concentration
of S will stop P becoming a boundary region and thus prevent head-end formation. It is also assumed that the acquisition of inhibitory properties is associated
with the synthesis of S when P becomes the head-end boundary value. These
points are illustrated in Fig. 1. It is very important to realize that the time for
determination will be very much affected both by the experimental conditions
and the nature of the assay. For example, true determination can only be
assayed by placing the piece under consideration in a position where S is at its
maximum value. Again, since the time for determination involves both a fall in
S and a rise in P, the conditions affecting the rate of fall of S must be taken into
account.
MATERIALS AND METHODS
All experiments were performed using Hydra Httoralis as described in Hicklin
et al. (1973) and Hicklin & Wolpert (1973) and were incubated at 26 °C. When
Dynamics of boundary regions in Hydra
501
Table 1. HI 2 onto regenerating 12...F
Results
Regenerating time
(h)
Number of
combinations
N
H
HF
1
2
3
4
5
6
20
24
14
10
23
12
20 (100 %)
23 (96 %)
13(93%)
7(70%)
0
0
0
1 (4 %)
1(7%)
3(30%)
16(70%)
12(100%)
0
0
0
0
7 (30 %)
0
Table 2. Regenerating 12 onto 12...F
Results
Regeneration time
(h)
Number of
combinations
N
H
HF
F
5
7
9
12
15
18
10
9
10
19
23
21
1 (10 %)
3(33%)
2 (20 %)
7(36%)
19(82%)
18(86%)
2 (20 %)
1(11%)
2 (20 %)
3(16%)
2(9%)
3(14%)
4 (40 %)
1(11%)
1 (10 %)
3(16%)
0
0
3 (30 %)
4(44%)
5 (50 %)
6 (32 %)
2 (9 %)
0
grafts were made onto regenerating animals, the healed end was carefully prised
open with pins. The regions of hydra are referred to by the H1234B56F terminology. The results are scored in terms of structures formed at the junctions
as described earlier. Very briefly, N signifies a normal animal with no structures
at the junction; H is head at the junction; HF is a head and a foot at junction;
F is foot at the junction.
EXPERIMENTAL RESULTS
Dynamics of region 1
Using lateral grafting, Webster & Wolpert (1966) showed that when the H is
removed, a new H is determined at the top of region 1 after about 4-6 h. Since
the combination 12/12...F nearly always gave structures at the junction whereas
H12/12...F was never observed to do this (Hicklin et al. 1973) one may ask how
long a regenerating 12...F could be left before an HI2 graft could no longer
inhibit head formation at the junction. After removing the head from prospective host animals, they were allowed to regenerate for up to 6 h, and a freshly
excised H12 region was grafted on each (Table 1). After 1-3 h of regeneration
before grafting, a very few animals formed distal structures at the junction;
those that did formed two or three tentacles only. However, none of the animals
which had been regenerating for 5 h could be inhibited when combined with an
H12 graft. In every case, distal structures, usually comprising an entire head,
502
J. HICKLIN AND OTHERS
Table 3. Head determination from region 1 in F...21/12...F
Source of graft
Region 1 from
F...21/12...F
Region 1 from
12...F
Time after
cutting/axial grafting
(h)
Number of
grafts
Number of positive
distal inductions
10
10
20
20
20
1 (10 %)
1 (10 %)
2(10%)
6(30%)
17(85%)
10
2(20%)
11
6(55%)
9
10
8 (89 %)
10(100%)
formed at the junction and a few animals formed a foot end as well. Feet were
not observed to form at the junction in animals which had been regenerating for
6 h before grafting, it is interesting to note that an estimate of the time required
for half of the animals to escape from inhibition by a H12 graft gives a figure of
about 4-5 h. This is similar to the time for determination of a region 1 using
lateral grafting (Webster & Wolpert, 1966) but in view of the different geometries
in the two assays this correspondence may be misleading.
The next experiment was designed to determine when the inhibitory properties of a H12 were acquired by a 12; how long must 12...F regenerate before the
12 region will behave like H12 when it is combined with 12...F? Animals were
cut at the top of region 1 to remove the head and allowed to regenerate for up to
18 h, by which time tentacle buds had formed at the distal end. At various times
after cutting, the 12 region was excised and combined with a freshly cut 12...F
host. It was found (Table 2) that about 15 h of regeneration were required prior
to transplantation before a substantial number of combinations did not form
structures at the junction. Even after 18 h of regeneration, three out of twentyone animals formed tentacles at the junction. It can be seen that the time of 15 h
obtained in the present experiment is considerably longer than the 4-6 h period
of regeneration necessary for head-end determination at the top of region 1,
which suggests that it takes some time for full inhibitory properties to be established at the distal end. This is a somewhat different conclusion from that which
Webster (1966#) arrived at concerning the re-establishment of inhibition along
the axis during regeneration; he found that a lateral graft of tissue from region 1
was normally absorbed when implanted into a 12...F animal which had been
regenerating for 4-6 h, whereas at earlier times the graft led to the formation of
a new head end. The probable explanation for the difference is that the relative
position of the tissues in his and our experiments is rather different.
In obtaining information concerning the dynamics of the gradients of
Dynamics of boundary regions in Hydra
503
positional value and that of the signalling substance, an important question is
whether during regeneration it makes a difference whether the regenerating
boundary is at a free cut end or not (Hicklin et ah 1973). Combinations consisting of F...21/12...F always form heads (usually two) at the junction (Hicklin
et al. 1973) and the time for head-end determination for region 1 from such
combinations was obtained by lateral grafting into region 2 of intact animals.
This time was compared with the time required by region 1 of regenerating
12...F. The results (Table 3) show that very few grafts taken from F...21/12...F
which had been regenerating for less than 6 h resulted in the formation of a new
head end in the host, whereas after the same incubation period, the distal tip
taken from the controls, regenerating 12...F, induced a head in most instances.
After 8 h of regeneration, however, by which time grafts from the region 1 of
regenerating 12...F all induced a head end upon transplantation, the majority of
grafts taken from regenerating F...21/12...F also appeared to be determined by
this time. Whereas just under 5 h of regeneration appeared to be sufficient for
half of the animals of the control series to become determined, for the experimental series a little more than 7 h of regeneration seem to be required. Head
determination appears to be faster at a free cut surface. It was interesting that
grafts taken from F...21/12...F often formed supernumerary tentacles and
occasionally two heads were formed following transplantation to a host animal.
This was never observed in the control series, where the inductions were always
of a single head, and suggests autonomy of the two 1 regions.
Mention must be made of two puzzling observations. First, that if 12...F is
allowed to regenerate for 18 h, by which time tentacle buds have appeared at the
distal end and then combined with a freshly excised 12...F, as in F...21/12...F,
head formation is not inhibited in the second part of the combination and two
rings of tentacles form. Secondly, if the 12...F is allowed to regenerate for 18 h,
and then a 1 region is grafted onto the distal end with the same polarity, head
formation is inhibited at the top of the graft region 1 and a ring of 12-15 tentacles
is formed at the junction. A foot does not form at the distal end of the graft
which remains for at least 8 days as a lump situated above the circle of tentacles.
Dynamics of region 3
As for region 1, the time for region 3 to become determined as a head end was
assayed by finding out how long 34...F can be left before distal structures will
form at the junction when it is combined with H12. At various times, a fresh
H12 was grafted to the regenerating end of the 34...F (Table 4). About 8-10 h
of regeneration are required for a regenerating 34...F to escape from inhibition
by an HI2 graft. This time should be compared with the 4-5 h required by
region 1. The result is consistent with the results of Webster & Wolpert (1966)
that the time for head-end determination increases with distance from the head
end.
It was of interest to compare the time of 10 h for the determination of region 3
32-2
504
J. HICKLIN AND OTHERS
Table 4. HI2 onto regenerating 34...F
Incubation
period
(h)
Number of
combinations
Results
N
H
HF
0
6
8
10
12
12
20
13
15
19
12(100%)
20(100%)
10(77%)
8(53%)
0
0
0
3(23%)
7(47%)
12(63%)
0
0
0
0
7(37%)
Table 5. Determination of head from region 3 using lateral grafting
Results
Inductions
Regeneration
(h)
Number of
transplants
0
6
10
15
20
10
15
17
12
10
(
Distal
0
0
6(35%)
10(83%)
10(100%)
Proximal
Graft absorbed
0
0
1?(6%)
0
0
10 (100 %)
15(100%)
10(59%)
2 (17 %)
0
Table 6. 12 onto regenerating 34...F
Results
Incubation period
GO
Number of
combinations
N
H
0
1
2
15
30
20
15(100%)
11(37%)
5 (25 %)
0
19(63%)
15(75%)
obtained by axial grafting with that required using lateral grafting, since Webster & Wolpert (1966) did not consider this region in their study. The procedure
employed was similar to theirs, except that half of the regenerating tip was
grafted at the end of the incubation period instead of the entire tip (Table 5).
Grafts taken from the regenerating tip of 34...F did not result in the formation
of any inductions when transplanted to the mid-gastric region of intact hosts
when taken from animals which had been regenerating for 6 h only. After 10 h
of regeneration, however, a number of the grafts elicited the formation of secondary axes consisting of hypostome and tentacles, although about 15 h of
regeneration were necessary before the majority of the 34...F had become
determined. The time for head-end determination may be estimated to be
11-12 h, which is similar to the value obtained by axial grafting. On one
Dynamics of boundary regions in Hydra
505
Table 7. Regenerating 3 onto 12...F
Results
Incubation
period
(h)
Number of
combinations
0
8
12
16
20
24
11
15
27
13
15
14
,
N
H
3 (28 %)
7 (47 %)
10(37%)
12(92%)
15(100%)
13(93%)
2(18: Vo)
5(33! Vo)
5(19! Vo)
0
0
0
HF
F
I
3 (27 %)
1 (7 %)
4(15%)
0
0
0
2(18%)
2(13%)
5(19%)
1 (8 %)
0
1 (7 %)
1(9%)
3(10%)
0
0
0
Table 8. Regenerating 3 onto 34...F
Results
ubation period
(h)
0
6
8
10
12
24
Number of
combinations
N
H
20
14
14
15
29
23
4(20%)
5 (36 %)
4(29%)
10(67%)
28 (97 %)
22(97%)
16(80%)
9(64%)
10(71%)
5 (33 %)
1 (3 %)
1 (3 %)
occasion, the graft from the regenerating distal tip of region 3 resulted in the
formation of an axis consisting of peduncle and basal disc; this suggests that
determined bud material may have been included as the graft. If so, it is possible
that the results may on other occasions have been affected by the proximity
of the budding region to the site of distal regeneration.
Grafts of 12/34...F do not form structures at the junction (Hicklin et al. 1973).
It is possible, therefore, to investigate how long regenerating 34...F may be left
before it can 'escape' from inhibition by a graft 12 region. 34...F regions were
excised from donor animals, incubated for a certain time and then a 12 region
was grafted on each. It was found (Table 6) that after as little as 1 h of regeneration, distal structures formed at the junction in well over half of the animals
when they were combined with a 12 region. This result is also of interest because
it shows that changes can occur quite rapidly. Moreover, if starvation delays
wound healing, it might provide an explanation as to why 12/34...F which have
been starved give heads at the junction (Hicklin et al. 1973).
The next experiment was undertaken to find out how long a period of regeneration 34...F requires before a graft of the region 3 behaved like a HI2
region in that it would inhibit the formation of structures at the junction when
combined with a 12...F host. It was found (Table 7) that between 12 and 16 h
of regeneration are required for about 50 % inhibition to be obtained. The time
for determination seems to be about 14 h. It should be noted that in none of
32-3
506
J. HICKLIN AND OTHERS
Table 9. Regenerating HI2 onto 12...F
Results
H12 incubation
period
(h)
Number of
combinations
1
2
3
5
7
20
22
30
24
20
N
F
20(100%)
22(100%)
20(67%)
6(25%)
0
0
10(33%)
18(75%)
17(85%)
3 05%)
the 0-h control series was the polarity of the graft reversed (compare table 8,
Hicklin et ah 1973).
In contrast to 12/34...F which gives no structures at the junction, 34/34...F
usually gives structures at the junction (Hicklin et al. 1973). How long does it
take for a 3 to become like a 12 such that when it is grafted to a 34...F no
structures form at the junction? This is similar to the previous experiment
except that acquisition of inhibition is being assayed against region 3 rather than
region 1 (Table 8). The results show that it requires about 8-10 h before a
region 3 can prevent head formation from 34...F. This is, as might be expected,
shorter than that required to inhibit 12...F as found in the previous experiment.
Changes at the proximal end
Investigations using axial grafting of the time required for changes in the
positional value of a region destined to become a foot-end boundary were
limited to one experimental situation only: more extensive study involving
lateral grafts is reported elsewhere (Hicklin & Wolpert, 1973). An investigation
was carried out to determine how long regenerating HI2 required to be left until
a foot was formed at the junction when it was combined as a graft with 12...F
(Table 9). The time required is between 3 and 5 h. This can be compared with
the 12 h required for foot-end determination by region 2, when assayed by
lateral grafting (Hicklin & Wolpert, 1973). Since the foot end exerts an inhibitory
influence on foot-end determination, this difference in times may be due to the
difference in the distance between the region being assayed and the foot end; in
the axial graft it is greater.
The effect of temperature
The effect of temperature on head-end determination was investigated using
lateral grafting of region 1 of regenerating 12...F (Table 10) and region 5 of
regenerating 56F (Table 11) at 26 °C and 16 °C. For region 1 the time was about
5 h at 26 °C and 11 h at 16 °C. For region 5 the corresponding times were about
16 h at 26 °C and 36 h at 16 °C. The <2i0 is thus about 2.
Dynamics of boundary regions in Hydra
507
Table 10. The effect of temperature on head-end determination of region 1
using lateral grafting
Regeneration
Temperature
(h)
No. of grafts
Absorbed
26 °C
0
2
3
4
5
6
7
8
6
10
12
16
18
24
36
48
20
15
20
20
15
12
15
10
20
20
40
20
24
12
10
11
19(95%)
13(86%)
17 (85 %)
14(70%)
7 (47 %)
4(33%)
2 (14 %)
1(10%)
18(90%)
18(90%)
9 (22 %)
3(15%)
1 (4 %)
1 (8 %)
16 °C
Head
induction
1 (5 %)
2 (14 %)
3 (15 %)
6 (30 %)
8 (53 %)
8 (66 %)
15(86%)
9 (90 %)
2(10%)
3(10%)
31 (78 %)
17(85%)
22(96%)
11(92%)
10(100%)
11(100%)
—
—
Table 11. The effect of temperature on head-end determination of region 5
using lateral grafting
Inductions
Regeneration
Temperature
(h)
No. of
grafts
26 °C
0
4
8
16 °C
>
Absorbed
H
F
10
20
15
3 (30 %)
5 (25 %)
5 (33 %)
7 (70 %)
15(75%)
10(69%)
12
20
.16(60%)
—
—
—
—
15
18
30
18
24
30
36
40
12
10
10
18
22
40
22
20
5 (42 %)
2(20%)
1(10%)
13(72%)
5 (22 %)
25 (62 %)
5 (23 %)
2(10%)
4(33%)
7 (70 %)
9 (90 %)
—
1 (5 %)
6(16%)
12(54%)
18(90%)
8 (40 %)
3 (25 %)
1 (10 %)
—
5 (28 %)
16(73%)
9 (22 %)
3(13%)
—
DISCUSSION
The main results are summarized in Table 12. The times for regions at the
boundary to become determined as a head end when assayed by grafting a HI2
on them are essentially the same as those found by lateral grafting. As reported
previously (Webster & Wolpert, 1966), the times increase with distance from
the head end. The times required by a boundary region to acquire the inhibitory
properties of a head end are longer than those required to acquire resistance to
508
J. HICKLIN AND OTHERS
Table 12. Summary of main results relating to changes at the
head-end boundary
Assay
Regenerating
Regenerating
Regenerating
Regenerating
Regenerating
Regenerating
12...F to resist inhibition by HI2
34...F to resist inhibition by HI2
34...F to resist inhibition by 12
12 to inhibit 12...F
3 to inhibit 12...F
3 to inhibit 3...F
Tirr.e (h)
4-6
8-10
1
12-15
12-16
8-10
inhibition. For example, a regenerating 12...F requires about 4-6 h to resist
inhibition, whereas 12-15 h is required for the 12 region to acquire the inhibitory
properties of an H12. This is a somewhat different conclusion from that drawn
by Webster (1966#) on the basis of lateral grafting: he found that the times for
region 1 to resist inhibition and the time for re-establishment of inhibition
following head-end removal were about the same. However, the assays used
were different.
One can attempt to interpret these results in terms of our current model
(Wolpert et al. 1974). This suggests when the positional signal falls a threshold
amount below P, P increases until it reaches the head-end boundary value. Until
this boundary value is reached, it is assumed that it is possible to inhibit further
increase in P if S is increased sufficiently. This has important implications for
our assays of head-end determination, for it emphasizes that both P and S must
be taken into account. Escape from inhibition need not imply an irreversible
determination, but simply that P is sufficiently greater than S, so that it continues
towards becoming a head end. This is particularly clear in the case where a
34...F can become resistant to inhibition by a 12 region in a rather short time.
In terms of the model, a head end acquiring inhibitory properties occurs only
when P has reached the boundary value. The suggestion is that the synthesis of
S lags behind that of P, and this could account for the longer times required for
the development of inhibitory properties. It is to be expected that, as the results
show, less time is required for region 3 to be able to inhibit a 3...F as compared
with a 12...F: the concentration of S required is less. The similarity in the times
required by region 12 and region 3 to inhibit 12...F is perhaps surprising, but
becomes less so if the variability in the results is taken into account, as well as
the extra distance that S must diffuse in region 12.
It cannot be too strongly emphasized that other criteria for determination,
even in terms of the framework of our type of model, are possible. We cannot,
for example, exclude a model in which irreversible determination of the head end
occurs when S falls a threshold amount below P. However, the effect of temperature suggests that this is not the case. A Q10 of 2 was found for head-end
determination and with H. attenuata a Q10 of 6 was reported (Wolpert et al.
Dynamics of boundary regions in Hydra
509
1972). This high dependence on temperature might be used to argue against the
time for determination being simply due to time for diffusion.
We do not yet have a quantitative model capable of accounting for all the
results but we are attempting to construct one. Our model (Wolpert et al. 1974)
draws quite heavily on the results obtained here. In fact, the results put severe
constraints on any simulation and we do not know if it is possible to construct
a simple model which will account for both the instantaneous grafts (Hicklin
et al. 1973) and the dynamics given here. It should also be remembered that the
quantitative data itself may not be consistent as different batches of hydra do
not always give comparable results. For example, our current culture requires a
longer time for a 34...F region to escape from a 12 region graft, than those
reported here, and 12/56F combinations do not give structures at the junction
as reported in Hicklin et al. 1973.
One factor that must be taken into account in the construction of any quantitative model is the effect of a cut surface on the changes in S and P. We have
found it difficult to make progress without assuming a significant leakage of S
from the cut surface. As pointed out in a previous paper (Hicklin et al. 1973)
regenerated heads usually form at surfaces that have been cut. The results on the
time for determination of a 1 region in a F...1/1...F graft provide direct confirmation for this since it takes significantly longer than when the 1 is at a free
end.
No explanation can be offered for the observation that fully determined head
ends, which already have tentacle buds, do not inhibit a region 1 but seem to
assimilate such regions into the regenerating head.
In this paper only changes at the boundary have been considered and in a
following paper the changes with time away from a boundary will be reported.
This work was supported by the Nuffield Foundation. We are indebted to Dr D. Summerbell for his constructive comments.
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