/ . Embryol. exp. Morph. Vol. 30, 3, pp. 727-740, 1973
Printed in Great Britain
727
Positional information and pattern regulation
in hydra: formation of the foot end
By J. HICKLIN 1 AND L. WOLPERT 1
From the Department of Biology as Applied to Medicine,
The Middlesex Hospital Medical School, London W.I
SUMMARY
The time required for foot end formation increases with distance from the foot end.
Using lateral grafting it was shown that both the peduncle and basal disc could induce a
proximal axis when grafted into the gastric region. The time for foot end determination
was shown to be about 4 h at the proximal end of the gastric region and to increase towards
the head end. In general the determination of the foot end is similar to that of the head end.
INTRODUCTION
Much less attention has been given to the development of the foot region
than the head region during pattern regulation in hydra (see Webster, 1971).
Our previous papers (Wolpert, Hicklin & Hornbruch, 1971; Hicklin, Hornbruch, Wolpert & Clarke, 1973) have also concentrated on the head end
boundary, and the model using gradients in positional signal and positional
value was primarily developed to account for the formation of the head end.
Boundary regions are of key importance in considering positional fields
(Wolpert, 1971) and it is essential to know whether the foot end boundary has
properties similar to the head end and what additional features must be added
to the model to account for formation of the foot end. The model for head
formation suggests that a new head will form when the concentration of a
diffusible substance S falls a threshold amount below its original concentration
which is represented by a non-diffusible parameter P. Since S is made at the
head end, and is constantly being destroyed, removal of the head will lead to
S falling and this leads to the formation of a new head. It would be very attractive if the interactions between S and P could also account for foot end formation: for example, might it not be possible that foot end formation was initiated
when the concentration of S rose a threshold amount above PI Unfortunately
this simple symmetrical model cannot easily be sustained since there are a
variety of grafts in which just the contrary is observed. For example, in grafts
of H12/12...F (the hydra is represented by H1234B56F as in previous papers)
1
Authors'address: Department of Biology as Applied to Medicine, The Middlesex Hospital
Medical School, London W1P 6DB, U.K.
47
EM B
30
728
J. HICKLIN AND L. WOLPERT
S will rise at the proximal end of the distal 2 region but no foot forms, whereas
in 12/12...F, when S is lower there, a foot often forms. From a variety of such
experiments we have concluded that a signal from the head end can inhibit
foot end formation.
There is good evidence that the presence of a foot can prevent the formation
of another foot in adjacent regions (MacWilliams & Kafatos, 1968). This ability
of the foot end to inhibit foot formation is of course analogous to the properties
of the head end and this analogy is further strengthened by our results (Hicklin
et al. 1973) which showed the symmetrical behaviour of H12/12...F and
H12...56/56F type grafts. It would seem that the simplest model to accommodate the results would be to postulate another gradient in a diffusible substance
S' which would be produced at the foot end. Foot formation would then depend
on S' falling to within a threshold amount of A—P, where A is a constant
greater than the maximum value of P. In addition S would tend to inhibit
head formation. It will be important to know if S' can also influence P. Is,
for example, the formation of regions 56, the peduncle, controlled by a positional
signal from the foot end ? The answer to these questions is not clear. If there
are two positional signals, what might be the relationship between them?
There is some evidence that the presence of a head inhibits foot formation
(Hicklin et al. 1973) and thus that a signal from the head may be dominant.
In this paper we investigate the factors controlling the formation of the foot
using the lateral grafting techniques employed by Webster (Webster & Wolpert,
1966; Webster, 1966) for the head end. In this, the dynamics of foot end formation are investigated by finding the time of foot determination as well as the
time at which foot end structures appear. When investigating determination
of the head end, grafts were taken from the tips of regenerating animals at
various times - if these resulted in the formation of a head end when grafted
laterally into host gastric regions (rather than being absorbed), a new head
end was considered to have been determined in the regenerating animal. The
time of determination of the head end was found to vary according to axial
position of the regenerating distal end. Here we use a similar technique for the
foot end.
MATERIALS AND METHODS
All experiments were performed using Hydra Uttoralis as described in Hicklin
et al. (1973) and carried out at 26 °C.
Lateral grafts were performed as described by Webster (Webster & Wolpert,
1966). Pieces of tissue were obtained from donor animals by removing transverse rings of tissue from selected body regions and cutting these into small
pieces. If the donor animal was regenerating, a small disc of tissue was taken
from the regenerating end, bisected, and one of the two pieces was used as the
graft. The graft was implanted in the body wall of the host in a small incision
at the selected site. Great care was taken to ensure that the endoderm of host
Formation of the foot end in hydra
729
and graft were in contact for correct healing, that the graft did not project
beyond the edge of the wound and that the size of the grafts was constant
throughout all these experiments.
Where it was important to minimize variation between time of cutting and
time of grafting, only small groups of animals (5-10) were used; the totals
in the tables therefore represent the pooled results of several experiments.
Experiments involving the transplantation of small amounts of stained hypostome and basal disc (Browne, 1909; Yao, 1945) have shown that where a
secondary axis is developed as a result of lateral grafting, the graft persists at
its distal end. It is thought that the presence of the graft influences cell movement and cell orientation in the host (Webster, 1971). In the present experiments
it was noted that the first change which indicated positive induction was a
localized swelling of the body wall, and became evident within 2 or 3 h after
grafting. The development of the outgrowth and its differentiation occupied
no more than 48 h. The secondary axes could be recognized as partial distal
or partial proximal. A secondary distal axis, the characteristic result of grafting
hypostomal tissue, usually consisted of a short outgrowth the diameter of which
was approximately equal to that of the distal gastric region of the host; it terminated in a head region (Fig. 1 a). Three other results have been described by
Browne (1909), Yao (1945) and Webster (Webster & Wolpert, 1966) in similar
experiments and some of these are illustrated in Fig. \(b,c). These were occasionally observed in the course of the experiments described here. The only
type which was not classed as a positive induction was Fig. l(c), where just
one or two tentacles formed. This was rare and with time the tentacles were
almost invariably absorbed. All the other types were characteristically stable
and were not absorbed.
A secondary proximal axis consisted of a peduncle and a basal disc, and it
was formed when pieces of tissue from either of these regions were grafted to
the gastric region of a host animal (Fig. 2). Occasionally, tissue grafted from
proximal regions apparently differentiated as a small basal disc in the side of
the animal. Such basal discs sometimes later disappeared and were not counted
as positive inductions.
It was noted that induced proximal axes did not appear to increase in size
after the first 48 h; the same appeared to be true for induced distal axes unless
the animals were fed. Fed animals with two heads became Y-shaped and then
V-shaped before splitting in two. On a very few occasions it was observed that
when the branch point of the two distal axes reached the budding zone, an
outgrowth developed on one of the distal axes close to the fork, and this outgrowth differentiated into a peduncle and basal disc. In these instances, the
separation occurred by cleavage of the isthmus of tissue which connected each set
of distal and proximal structures.
One interesting phenomenon observed was the speed at which an induced
proximal axis would migrate down the gastric region, traversing perhaps its
4-7-2
Fig. 1 A and B. For legend see facing page.
I—I
H
tn
o
r
o
r
Formation of the foot end in hydra
731
Fig. 1. Examples of induced secondary distal axes following grafting (arrow). (A)
Short axis with hypostome and tentacles. (B) Short axis with long single tentacle.
(C) Single tentacle.
entire length in a matter of a few days. However, once a proximal axis had
reached the lower limit of the budding zone it moved only very slowly, gradually
fusing with the host's peduncle over the period of a week or so. Distal inductions remained at the distal end of the animal and appeared to compete with the
host head for control of the axis.
RESULTS AND DISCUSSION
Time required for different regions to form basal disc
A simple cutting experiment was first of all performed to investigate the
time taken for foot regeneration in animals cut at different regions. The formation of a basal disc at the proximal end was gauged by testing for the known
adhesive properties of the basal disc region: the dish containing the animals
was agitated for a few seconds and if an animal remained attached to the bottom
it was judged to possess a basal disc. Meticulous care was taken to keep the
732
J. H I C K L I N AND L. W O L P E R T
Fig. 2. Induced secondary proximal axis (arrow)
Table 1. Times for basal disc regeneration at different levels
Total with basal disc after:
Regions
HI
H12
H1234
H1234B
H1234B5
No of
animals
24 h
48 h
72 h
96 h
30
20
20
26
20
0
0
7(35%)
0
8 (40%)
2 (7%)
15 (75%)
20(100%)
2 (8%)
20(100%)
4(13%)
17(85%)
—
4(15%)
—
7 (23%)
20(100%)
—
6 (23%)
—
dishes free from any debris which might prevent it from attaching to the dish.
Any detached buds were removed.
The results (Table 1) show differences in the time required to form a basal
disc. In the gastric region these times appear to be graded up the axis while
basal disc formation occurs much faster from the 5 region than from the 2
733
Formation of the foot end in hydra
Table 2. Lateral grafts of proximal regions
Results
A
Source
of graft
tissue
Region to which
transplanted in
host
No. of
transplants
F
F
F
H
Middle 1234
Middle 56
15
27
13
5
6
5
Middle 1234
Middle 1234
Middle 56
16
26
25
Proximal
axis
induced
0
20(74%)
0
13(81%)
17 (65%)
0
Graft
absorbed
15(100%)
7 (26%)
13(100%)
(foot remains)
3 (19%)
9 (35%)
25(100%)
region. HI regions formed basal discs very slowly, and most had not done so
even after several weeks. It is interesting that H1234B animals which had been
cut just under the youngest bud took as long to regenerate the basal disc as
did HI regions. Results from H1234 animals were similar to those from H1234B5
animals: by 24-48 h they had all regenerated a basal disc.
Regeneration of the peduncle and basal disc in animals cut across the gastric
region (i.e. HI, H12, H1234) was marked by a series of visible changes whose
duration appeared to vary according to the axial position of the regenerating
end. There was first a noticeable narrowing of the most proximal end, which
in the H1234 animals was evident as early as 4 or 5 h after isolation. This narrowed zone then began to acquire the pale translucent appearance characteristic
of peduncle tissue. At a later stage the basal disc could be distinguished at the
proximal end, though it was not until the basal disc had visibly differentiated
that the peduncle assumed quite its normal form and proportions, having
until then appeared somewhat shorter and broader than normal.
Although H1234B regions also appeared to follow this sequence of morphological changes, in most instances the animals remained in the first stage for
several days without regenerating a basal disc. After 7 days all had regenerated.
Neither bud initiation nor bud detachment appeared to be affected during
regeneration in these animals.
Lateral grafts of peduncle and basal disc
No reports are known in which it is shown that region 56, the peduncle, is
capable of giving rise to a new foot end following lateral transplantation into
another animal. However, it is well known that the outcome of any grafting
operation depends on (a) the source of the graft and (b) the site to which the
graft is transplanted in the host. For example, Webster (1966) found that a
graft taken from region 1 was absorbed when transplanted into the midgastric
734
J. HICKLIN AND L. WOLPERT
region of an intact animal, but resulted in the formation of a head end when
transplanted to its foot.
The following experiment investigated the behaviour of peduncle and basal
disc grafts transplanted to different positions on the host axis (Table 2).
Pieces of tissue of approximately the same size were isolated from three
positions on the linear axis of donor animals: from the foot (about one quarter
of the basal disc) and from the top of regions 5 and 6. Grafts were then transplanted laterally into hosts. Grafting basal discs was particularly difficult
because this region is so sticky; also the thick ectoderm caused some grafts to
roll up and enclose the endoderm, the cells of which are relatively sparse in
this part of the animal. It is suspected that this rolling-up may have affected the
success of the grafting operations in a few instances.
Grafts of basal disc were always found to be absorbed when transplanted to
a host's head region; occasionally the graft passed into an elongating tentacle
where it was not absorbed, but was lost from the tip within a few days. Basal
disc grafts to the gastric region resulted in the formation of a proximal axis
(i.e. consisting of a peduncle and basal disc) in 20 out of 27 animals. From its
earliest stages the induced axis had the morphological appearance of peduncle
tissue and within 24 h a basal disc of normal size was evident at its tip. When the
peduncle was chosen as host site, the foot graft did not induce an axis but
migrated to the basal disc as a small sticky patch. Once this migration had been
accomplished, the graft disappeared, having fused with the glandular surface
of the disc.
Grafts from regions 5 and 6 to the midgastric region of hosts resulted in a
high proportion of proximal axes forming. Peduncle tissue grafted into the
gastric region resulted in the formation of an outgrowth which retained the
dense texture and pink colour of gastric region tissue for the first 24 h or so
after grafting. Between 24 and 48 h after grafting this outgrowth became pale
and translucent and a basal disc formed at its tip. When the graft was taken
from region 6, the basal disc seemed to form a little sooner than when the graft
was from region 5. Grafts of peduncle (region 5) into the peduncle of hosts
were invariably absorbed.
In one animal (not in Table 2) a graft into the head region healed into the
subhypostomal region of the host, resulting in the formation of a tiny axis
consisting of a peduncle and basal disc - it was not clear whether this was an
induction or whether part of the graft had reorganized into peduncle.
The most important point to emerge from these results is that both peduncle
and basal disc appear to be able to bring about the formation of a secondary
proximal axis when transplanted to the midgastric region of a host animal.
The finding that peduncle tissue can induce seems to conflict with previous
reports concerning the behaviour of grafts taken from this region. It may
represent a species difference. The very high proportion of proximal inductions
which resulted from grafting basal disc into the midgastric region suggest that
735
Formation of the foot end in hydra
Table 3. Time of determination of foot end at different levels
Time after
cutting
Donor
(h)
H1234
1
2
3
4
5
12
15
1-5
2
3
5
7-5
9
14
7-5
9
11
13
15
18
24
5
12
18
24
36
48
72
H123
H12
HI
No. of
transplants
Proximal
induction
Absorbed
18
20
32
34
24
21
15
15
22
23
24
16
15
13
24
21
22
17
18
22
14
11
22
26
30
30
30
31
1 (6%)
1 (5%)
16 (50%)*
23 (68%)*
22 (92%)*
19 (90%)*
15 (100%)*
0
1 (5%)
1 (4%)
1 (4%)
5 (31%)
13 (87%)
13 (100%)
2 (8%)
5 (24%)
8 (36%)
13 (76%)
14 (78%)
21 (95%)
14(100%)
0
3 (14%)
4 (15%)
1 (3%)
1 (3%)
1 (3%)
0
17(94%)
19(95%)
16(50%)
11(32%)
1+JF(8%)
1 + 1F (5 % )
0
13 + 2F(100%)
20+IF (95%)
21 + IF (96%)
20 + 3F(96%)
9 + 2F(69%)
2(13%)
0
22(92%)
13 + 3F(76%)
12 + 2F(64%)
3 + lF(24%)
4(22%)
1 (5%)
0
11(100%)
18 +IF (86%)
21+ IF (85%)
28 +IF (97%)
29(97%)
29(97%)
31 (100%)
* In these there were a few cases of both proximal and distal axes occurring
together.
this is a standard result. The technical difficulty of grafting fragments of basal
disc could well account for the finding that seven of the grafts were absorbed.
On the basis of these results alone, it is not possible to differentiate between
the behaviour of grafts taken from the peduncle and those from the basal
disc. There was, however, a difference when grafts of peduncle and of foot were
transplanted to the mid-peduncle of host animals. No inductions were formed
as a result of grafting either of these regions, but peduncle grafts were completely absorbed while foot grafts appeared to be stable until lost by attrition.
This confirms the finding of MacWilliams & Kafatos (1968) that a grafted
basal disc will inhibit the formation of another disc in its neighbourhood (see
also MacWilliams, Kafatos & Bossert, 1970).
736
J. HICKLIN AND L. WOLPERT
Table 4. Determination of head end from distal region end 5
Results
Inductions
Regeneration
(h)
No. of
transplants
0
6
12
18
30
15
10
29
10
10
Proximal
No
induction
10(67%)
7(70%)
12(41%)
1 00%)
0
5(33%)
3 (30%)
17(59%)
2(20%)
1 (10%)
A
r
Distal
0
0
0
7(70%)
9(90%)
Time of determination of proximal regions
The above experiment seems to indicate that both peduncle and basal disc
can elicit the formation of a secondary proximal axis consisting of peduncle
and basal disc when transplanted into the midgastric region of an intact host
animal. These results do not, however, resolve the question whether peduncle
specifically can induce a proximal axis without first having been determined as
a foot end. The test also provides a means of investigating the time at which a
regenerating proximal end is first capable of inducing proximal structures
when grafted laterally into a host animal, which may be taken to be the time at
which the new foot end is determined. We have investigated the time of
determination of the proximal regions in regenerating HI, HI 2, HI23, and
HI234 tested by lateral grafting into the midgastric region (regions 2 and 3)
of intact host animals.
Donor animals were cut to provide HI, H12, H123, and H1234 pieces and
at various times after cutting the proximal tip of the regenerating animal was
removed, split into two pieces and one of these pieces was then transplanted
laterally into the midgastric region of an intact host animal. All grafts were
made before the animals had regenerated a basal disc.
As might be expected the results (Table 3) show differences in the time at
which proximal structures are determined in the four groups of regenerating
animals. From the results, estimates were made of the approximate time after
which 50 % of the grafts belonging to each series induced proximal axes.
This will be referred to as the T50 for foot determination. For the H1234 this
was about 4 h, for the HI23 about 8 h, and for HI2 about 12 h. These results
indicate an axial gradient for proximal end determination, which may be analogous to that for head end determination (Webster & Wolpert, 1966).
Occasionally, distal axes and sometimes mixed distal and proximal axes
were obtained when the proximal end of a regenerating H1234 region was
transplanted, but the frequency of such inductions diminished with longer
regeneration periods. This result is probably due to the inclusion of determined
131
Formation of the foot end in hydra
Table 5. Effect of presence of head end in hosts to which lateral grafts are
made
Results
Level of
donor from Level to which
which graft transplanted in
taken
host
Mid-gastric
Mid-gastric
F
F
Mid-gastric
Mid-gastric
Host's
head
present
Yes
No
Yes
No
Yes
No
Inductions
No. of
transplants
24
24
18
20
26
26
Distal
Proximal
Graft
absorbed
0
5 (21 %)
2(11%)
7(35%)
0
0
1 (4%)
1 (4%)
0
0
13(50%)
21 (81%)
23(96%)
18(75%)
16(89%)
13(65%)
13(50%)
5 09%)
bud material in the 4 region (Sanyal, 1966). Where mixed inductions were
obtained they have been included as proximal inductions in Table 3. In a
small number of animals belonging to each series the graft only appeared to
differentiate into a basal disc. These have been recorded in Table 3 as having
been absorbed. Although these could be classed separately, because of their
rareness this was not done.
Determination of the head end from the distal end of region 5 {the peduncle)
Webster & Wolpert (1966) showed by lateral grafting that a new head end
became determined at the top of region 5 in regenerating 56F about 18 h after
cutting. The results in Table 2 indicate that a graft taken from the distal end
of region 5 can induce a peduncle and basal disc when immediately grafted
into the gastric region of a host animal. There will therefore presumably be a
period when, if tested by lateral grafting, regenerating 5 tissue can be said to be
undergoing a change in properties. This may be shown either by a period when
the inductions are predominantly both distal and proximal or a period when foot
end inductions have ceased and head end inductions have not yet started. An
experiment was performed to investigate these possibilities.
Animals were cut at the top of region 5 and at various times after cutting
the regenerating top was removed, bisected and half was then implanted into
the gastric region of an intact host animal. It was found (Table 4) that grafts
taken from the distal tip of 56F which had been regenerating for less than 6 h
formed a high proportion of proximal axes consisting of peduncle and basal
disc. After 12 h of regeneration, fewer proximal axes resulted but in a number of
instances the graft differentiated either into a basal disc or into one or two tentacles which were eventually absorbed. After 18 h of regeneration, distal axes
predominated in grafting tests, a finding which accords with Webster & Wolpert's (1966) results.
738
J. HICKLIN AND L. WOLPERT
These results suggest that the switchover point from foot-like to head-like
transplantation properties occurs at the distal end of an isolated 56F region
after about 12 h of regeneration.
Effect of the presence of the head on the formation of proximal regions
A question of considerable theoretical significance is whether the presence
of the head affects the regeneration of proximal regions. Previous attempts
to investigate this have in general been unsatisfactory, relying as they have done
solely on the reappearance of the basal disc at the cut end (Mookerjee &
Bhattarcharjee, 1967). We (Hicklin et al. 1973) have suggested that the head
end may inhibit foot formation. We have further investigated this by comparing
the behaviour of grafts placed in intact hosts with those placed in hosts from
which the head had been removed.
Pieces of tissue were isolated from regions 3 and regions 5. In one series,
region 3 grafts were transplanted to the midgastric region of host animals,
and in another to the foot; region 5 grafts were transplanted to the midgastric
region of hosts. Immediately after each grafting operation the host's head
region was removed by cutting at the top of region 7. Control experiments
using intact animals were run for each series (Table 5).
The results of grafting region 5 give some indication that the presence of a
head region may have an inhibitory effect on the development of a foot end.
Region 5 grafts transplanted into decapitated animals resulted in a significantly
greater number of proximal axes forming than in the intact control group
(P less than 0-05 using x2 test). No such difference appeared when grafts
were taken from region 3 and transplanted to the midgastric region of host
animals; instances of induction were rare anyway. Grafts of region 3 to the foot
sometimes resulted in the formation of a tiny secondary distal axis, and though
the proportion of animals giving this result was slightly higher in the decapitated
group than in the control series the difference is not significant. The development of these secondary axes was peculiar in that, unlike grafts of head region
to the gastric region, an outgrowth formed which only later developed tentacles
at its distal end. In this respect it resembled bud development, except that the
induced distal axis did not separate from the host, at least for several days.
This formation of a secondary axis in the peduncle as a result of grafting
gastric region into it extends Webster's (1966) finding. In this, a graft of region 1
when transplanted to the foot was found to result in a secondary axis with hypostome and tentacles whether or not the host was regenerating a head at its
distal end.
Formation of the foot end in hydra
739
DISCUSSION
The interesting point to emerge from these experiments is that determination
and development of the foot end appears to be symmetrical with that at the
head end. Like grafts from the head end, lateral grafts from the foot end are
capable of 'inducing' or organizing a secondary axis. This property is also
shown by peduncle tissue grafted to the gastric region though not to the peduncle.
This is a new finding.
The times for foot regeneration and determination are also similar to those
for the head end (see Webster & Wolpert, 1966). Firstly there is a gradient
in the time required for the development of the foot after cutting, depending
on the distance of the regenerating end from the foot. A foot will form only
very slowly from the cut proximal end of HI. This is comparable to the slow
formation of the head in 6F animals (Webster & Wolpert, 1966). There is
also a gradient in the time for foot end determination as assayed by lateral
grafting. The times are short and determination rapid. For peduncle tissue it
is not even possible to measure a time for determination since it will induce
immediately following lateral grafting, suggesting that determination takes
place within 1 h. For region 4 the determination time is only 3-4 h. For head
determination, the time required by the 3 region is about 10 h (Hicklin &
Wolpert, in preparation) and a similar time is required for foot determination.
In view of the ability of region 5 to ' induce' a proximal axis when grafted into the
gastric region it was interesting to observe the behaviour of a 5 region when it
was regenerating the head: there was a switch from proximal to distal inductions between 12 and 18 h. It is perhaps surprising that the ability to induce
proximal axes had not been lost even after 12 h of regeneration.
We have obtained some further evidence that the presence of the head tends
to inhibit foot formation which supports our previous conclusion (Hicklin
et al. 1973). A higher percentage of proximal inductions was obtained with
grafts from region 5 when the host's head was removed than when it was present.
These similarities of behaviour exhibited by the foot and head confirm our
view that they are the boundary regions of a bipolar positional field. These
boundary regions appear capable of organizing a new axis, presumably by
establishing a new boundary to the positional field. The head end may be dominant in so far as it may inhibit foot end formation. One may account for foot
end formation in a similar way to head end formation: their symmetrical behaviour with respect to axial grafting has already been established (Hicklin
et al. 1973). What is far from clear is whether they contribute to the assignment
of positional information to the intermediate regions and if so what their
relative contribution may be.
This work is supported by The Nuffield Foundation.
740
J. HICKLIN AND L. WOLPERT
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{Received 16 April 1973)