/. Embryol. cxp. Morph. Vol. 30, 3, pp. 701-725, 1973
701
Printed in Great Britain
Positional information and
pattern regulation in hydra: the formation of
houndary regions following axial grafts
By J. HICKL1N, 1 A. HORNBRUCH, 1 L. WOLPERT 1 AND
M. CLARKE
From the Department of Biology as Applied to Medicine, The Middlesex
Hospital Medical School, London W.I,
and Institute of Computer Sciences, University of London
SUMMARY
A two-gradient model is proposed for regulation and regeneration of the head end of
hydra in terms of positional information. One gradient is set up by a diffusible substance
made at the head end, which may be regarded as a positional signal, and the other is a
more stable cellular parameter which is the positional value. The rule for head end formation is that the concentration of the diffusible substance falls a threshold amount below the
positional value. A variety of instantaneous axial grafts have been carried out which are
discussed in terms of this model. Some simple computer simulations of diffusion are provided. There is some evidence that the source of the positional signal is localized at the
head end. Some experiments on changes in polarity are given. It also seems that the positional
signal from the head end inhibits foot end formation.
INTRODUCTION
Regeneration and pattern regulation in hydra can be considered in terms of
a positional field (Wolpert, Hicklin & Hornbruch, 1971) with the hypostome
or head end, and basal disc or foot end as the boundary or reference regions.
A wide variety of grafting experiments have been carried out using the development of the head and foot regions as markers (Webster, 1971). From these
grafting experiments various models have been put forward to account for
the results. Our current model is framed in terms of positional information
(Wolpert, 1969, 1971) which may provide a much more general, and possibly
universal, framework. The basic idea is that pattern formation is essentially
a two-step process: first the assignment of positional information and subsequently the interpretation of this positional information by the cells leadmg
to molecular differentiation. Regeneration of hydra is, in these terms, the
1
Authors'1 address: Department of Biology as Applied to Medicine, The Middlesex
Hospital Medical School, London W1P 6DB, U.K.
702
J. HICKLIN AND OTHERS
Source
H1 2 3 4 B
56
F
Fig. 1. Diagram to illustrate the postulated changes in S and P during normal
regeneration. In the intact animal (a) the concentration of S isfixedat 100 at the head
end and at 10 at the foot end. When the head is removed, the concentration of
S falls; (b) when it reaches a threshold value below P (about 1-5 h) P is synthesized
locally until it is back to the original boundary value of 100 (about 4 h); (c) S is then
made so that its concentration at the head end is once made a 100 (about 8 h);
(d) the mechanism whereby P increases away from the boundary is slow and
may be set by P following S.
reassignment of positional information when the system is perturbed: that is
the re-establishment of boundary values and the form of the gradient.
The original experiments on the factors controlling hypostome formation
performed using lateral grafts (Webster & Wolpert, 1966; Webster, \966a,b),
suggested a model in which there were two gradients which decreased from the
head end towards the basal disc. One was a gradient in an inhibitor of head
formation, and the other was a gradient in threshold of inhibition. The inhibitor
was considered to be made at the head end. The rule for head-end formation
was that the concentration of inhibitor should fall below that of the threshold.
In a more recent formulation (Wolpert, Hornbruch & Clarke, 1973) in terms
of positional information, the inhibitor gradient is represented by a diffusible
substance S, the positional signal, which is made by a source at the head end,
the gradient being established either by the presence of a sink at the foot end,
or by continuous breakdown throughout the animal. The other gradient should
be thought of as a cell state, the positional value, P, which can, for convenience,
Boundary regions in hydra
703
be represented by the concentration of a non-diffusible substance. It is a more
stable cell property and is equivalent to the threshold of the original model.
Its concentration is changed by synthesis and breakdown of P according to
certain rules. The mechanism whereby the value of P is determined is not clear
but one possibility is that it is specified by S by a follow-up-servo type of
mechanism (Wolpert, 1971). Another type of mechanism is based on active
polar transport (Webster, 1971). The rule for making a head end boundary is
that 51 falls a threshold amount below P. Thus, in normal regeneration, removal
of the head end removes the source of S, and because S diffuses and is being
destroyed, its concentration will fall most rapidly at the head end (Fig. 1).
When S reaches a critical value below P, P increases at that point to the
boundary value and at that time becomes a new source of S. More detailed
dynamics of the model will be considered in later papers. However it is worth
mentioning that we have recently obtained evidence that the transmission of a
signal from the head end preventing head formation in adjacent regions is consistent with a model based upon diffusion (Wolpert, Clarke & Hornbruch, 1972).
The model is essentially a synthetic one and may be regarded as a convenient
current redescription of the numerous grafting experiments. It might even
give some insight into the mechanisms. We are very conscious that other simple
models might also account for the results, but we have been unable to think of
them. The following features of the present model should be kept in mind when
considering the results: (i) there are two gradients but only one involves a
diffusible substance, (ii) the changes in positional value are by synthesis and
destruction, (iii) polarity is determined by the dynamics of the gradients, (iv)
the gradients are inferred from head and foot end formation but may be assumed to provide positional information, (v) the hydra is treated as if it were
made up of a single-cell type and the distribution of the various cell types is
ignored (see Bode et al. 1973), (vi) cell growth and division play no role (Wolpert
et al. 1971; Hicklin & Wolpert, 19736).
As we have pointed out (Wolpert et al. 1971), grafting provides a powerful
but limited technique for investigating positional information in hydra. It is
powerful because it is easy to do a very large variety of grafts and restricted
because of the absence of good markers along the axis which can be used to score
the results. Our most reliable markers in hydra are the head and the foot,
and it is the presence or absence of these structures in graft combinations from
which we infer the properties of the gradients.
In this paper we are primarily concerned with what happens when various
instantaneous axial grafts are made. Such instantaneous grafts, which are made
immediately after cutting, are distinguished from those in which changes with
time are investigated, and grafts are made at various times after or before
operative procedures. That will be the subject of later papers. The interest in
instantaneous axial grafts relates to whether or not heads form at the junctions
between grafts according to the model: that is, whether a head end will form
704
J. H I C K L I N AND OTHERS
Fig. 2. Photograph of hydra to show the regions H1234B56F.
when S falls significantly below P. To assist the analysis some simple computerbased simulations of diffusion within the grafts have been carried out.
Attention will be focused here on the head end rather than the foot end, which
will be considered in another paper (Hicklin & Wolpert, 1973a). It seems
that the formation of the foot end might be viewed in terms of an analogous
system which is a mirror image of that for the head end. For example, there
would be a diffusible substance S' produced by the foot end, and foot end
formation would depend on S' falling a certain distance below P' (which might
be the inverse of P). In addition, it seems that S tends to inhibit foot end formation.
We have a convenient notation for the regions of the axis in hydra (Fig. 2).
The head region, consisting of hypostome and tentacles is represented by H,
and the digestive zone by four equal-sized regions, 1, 2, 3, 4; B designates the
budding region; 5 and 6 the distal and proximal halves of the peduncle, and
F the foot or basal disc. In terms of this notation a graft of head and distal
gastric region onto the subhypostome of another animal is given as H12/12.. .F.
Boundary regions in hydra
705
Fig. 3. Illustration of Type H - head at the junction which is indicated by an arrow.
MATERIAL AND METHODS
Hydra littoralis was the species used in all experiments. The animals were
grown from a clone originally supplied by Dr H. M. LenhofT. The culture
methods were as described by Webster (Webster & Wolpert, 1966). By regularly
cleaning the culture dishes, fungal and protozoan infections were kept to a
minimum; antibiotics and fungicides were not normally employed. Experiments were usually begun 18-20 h after feeding, using adult animals with one or
two buds. Starved animals were used for a small number of experiments. All
operations were carried out on a plasticine surface under a culture medium
('M' solution). Animals were always left to recover their maximum length
before cutting. Grafted animals were transferred to watch-glasses and placed
in an incubator at 26° C. Their medium was changed every day, but they were
not fed.
Axial grafting was performed as follows: the graft was obtained from a
donor animal by cutting between the regions indicated in Fig. 2. Next a host
animal of approximately the same size as the donor was sectioned at a selected
level and then both graft and host pieces were threaded with the desired polarity
onto a human hair. Host and graft were kept in contact by floating the hair
on the surface of the operating dish. They were left to heal together for 6090 min, after which the hair was removed and the combination transferred to
a watch-glass for incubation at 26 °C.
706
J. H I C K L I N AND OTHERS
Fig. 4. Type F - a foot forms at the junction between the grafts.
The essential advantage of axial grafting over lateral grafting is that it
allows the experimenter to control the orientation of the graft with respect
to the host, and therefore its polarity. Although duplicated batches of transplanted animals exhibited the same pattern of results, it was very common
to find that within a single experiment individual results often differed considerably; two animals subjected to what appears to be an identical transplantation operation may develop quite differently. Moreover, over many
months the cultures may change quite significantly.
The results of the axial grafts were assessed after 48 h for the formation of
head and foot structures at the junction and at any cut end. The following were
the main classes of results.
Normal (type N). The axis remained linear (with a head at the distal end)
and no structures were formed at the junction. Animals which were particularly
long appeared to regulate their length by continuing to bud for a longer period
than similarly starved control animals (Webster & Hamilton, 1972). All E12\
12...Ffa\\ into this class.
Boundary regions in hydra
707
Fig. 5. Type R - a foot has formed at the distal end {arrow).
Head at junction {type H) (Fig. 3). A head at the distal end and usually a
single head at the junction but including occasional animals forming two or
more heads or just one or two tentacles. None of these animals was observed
to separate into two individuals. Sometimes when an entire head region formed
at the junction, it eventually migrated and merged with the distal head; when
only a few tentacles were formed, they tended to migrate down the gastric
region and were absorbed, though sometimes they became shortened and sticky
at their tips like a tiny peduncle and basal disc. Budding in type H animals
appeared to be reduced if more than one full head region had formed at the
junction.
Foot at junction {type F) (Fig. 4). A head at the distal end and proximal
structures at the junction consisting of usually one (but often two) short
penduncles terminating in a basal disc. Sometimes no secondary axis formed,
but a basal disc developed, and the main axis assumed the translucent appearance characteristic of peduncle tissue. Very occasionally the combination
separated into two pieces, resulting in one more or less normal individual and
one with a foot at both its distal and proximal ends. The usual later development of F-type animals, however, was that the entire host axis became consumed by buds between its junction with the graft and its own peduncle. This
Y-shaped animal often returned to a linear shape by loss of one peduncle
(often the host's original one) or by attrition of the foot end of each. Curiously,
differentiation of peduncle tissue appeared to occur in disto-proximal direction
708
J. HICKLIN AND OTHERS
H
1
2
3
4
irTT3|4|5|6|7|g|»|lO
100
o
U
10
Level at which head is removed
100 -
90 -
80 -
70 -
60 -
50 -
Fig. 6(«). Diagram of a 17-celled hydra to show the system used for computer simulation of changes in S; and (b) the result on the concentration of S of removing the
head (3 different time intervals after head removal).
Boundary regions in hydra
709
90 -
80 -
«
7 0 -
60 -
50 T
1
T
2
T
3
Fig. 7. The distribution of S 4 h after forming a 72/72... F graft (•).
D, S at the time of grafting.
from the graft junction 48 h after the combination had been made, and in some
instances appeared to involve a considerable part of the host gastric region.
Foot and head at junction (type FH). In these animals the two types of structure at the junction appeared either at different levels on opposite sides of the
long axis, or one immediately above the other on the same side. Foot end
structures were always situated more distally at the junction than head end
structures. This suggests that although the graft and host had healed together,
they maintained their original polarity and regulated independently. Sometimes
graft and host separated after some days when the link between them had
become a second peduncle for the 'graft' individual. It should be noted that
the last bud forming in type F animals sometimes failed to detach; the animal
then presented a similar appearance to type FH animals.
The following two classifications are principally according to the type of
structures forming at the regenerating distal end of combinations and were
obtained only rarely.
Reversal of polarity (type R) (Fig. 5). These animals formed a foot at the
distal end of the graft indicating a reversal of its polarity. All 56/12...F fall
into this class. Usually one or more heads were formed at the junction between
graft and host; occasionally, however, head formation was inhibited, resulting
in a spindle-shaped animal.
Inhibition of graft (type I). No obvious structures formed at the distal end
of the graft and we infer that it was inhibited. Buds occasionally developed
in the graft. Either head or foot end structures formed at the junction between
graft and host.
710
J. HICKLIN AND OTHERS
3
Time (h)
4
5
90e H12/12
80 •
H12/12...F
{a)
H123/12. . .F
•2 7 0 -
12112.. . . F
H1234112...
F
60-
H12/56F
50-
12156
12J56F
Fig. 8. (a) Changes in S with time at distal end of region 1 at the junction in grafts
of the type H12\12...F. (b) Change in S with time at the proximal end of region 2
at the junction in grafts of the type HI2156...F.
Computer simulation of changes in S
It is difficult to be intuitive about the changes in the postulated diffusible
substance, S, in various graft combinations. We have therefore carried out
some simple simulations without any attempt to simulate the formation of new
boundary regions. We have used a hypothetical hydra 3 mm long made up of
17 cells. All regions except the foot are assigned two cells (Fig. 6a). The source
of S is at the head in cell 1 and is maintained at a constant concentration of
100. S is destroyed by a sink at the foot end, cell 17, where the concentration
is maintained at 10. The changes in S were determined from the diffusion equations using the well known Crank-Nicholson procedure. The diffusion constant
was taken as 2-00 x 10"7 cm2/sec (Wolpert et ah 1972).
It should be emphasized that the absolute values of S will be quite sensitive
to the length of the hydra and the relative proportions of the different regions.
The proportions we have chosen are no more than reasonable and thus the
results should only be taken as indicating changes in a semi-quantitative man-
Boundary regions in hydra
711
90-
Oh
70-
g 50-
30
5
6
1
2
3
4
B
Fig. 9. Changes in concentration of S following formation of a 56\12...F graft.
ner. In these simple simulations, changes in P are not taken into account and
the P value in any cell is equivalent to the S value in the intact animal.
The effect on the distribution of S following removal of the head region, the
source of S, is shown in Fig. 6(b). It can be seen that the amount S falls, increases
with time. Fig. 7 shows the distribution of S 4 h after a 12/12...F graft. The
changes in S at the 1 region of the host (or proximal region) with time, for a
variety of combinations of the type H12J12...F is shown in Fig. 8(a). Fig. 8(6)
shows the changes with time at the proximal end of the 2 region in HJ2/56F
and related grafts. Finally, Fig. 9 shows the changes in S in a 56/12...Fgraft.
These examples provide some information on the way that S will change with
a variety of graft combinations. An interesting feature is that where there are
large initial differences in the concentration of S, the main changes occur within
the first hour after grafting.
EXPERIMENTAL RESULTS AND DISCUSSION
Grafts of distal parts of axis to region 1 of host
In this series of combinations, grafts of part of region HI234 (with or without
the H) were combined with hosts which had been cut at the top of region 1.
The experiments and their results are summarized in Table 1. For some of these
combinations computer simulation of the changes in S following grafting has
been performed (Figs. 7, 8 a).
The combination H12j34...F'\s a control for the effect of grafting and never
46
E M B 30
712
J. HICKLIN AND OTHERS
Table 1. Grafts of distal regions onto region 1
Composition
Graft
H12
H12
H123
H123*
H123
2
11
12
Results
Host
No.
34...F
12...F
12. ..F
12...F
20
20
21
11
16
17
30
24
12
12...F
12. ..F
12
N
HF
0
20(100%)
0
20(100%)
11 (52%)
1 (5%)
4 (36%)
4 (36%)
0
16(100%)
3 (18%)
5(29%)
4 (13%)
15(50%)
8(33%)
3 (13%)
* These included some of the 4 region.
F
H
0
0
0
0
0
9(53%)
7(23%)
8(33%)
0
0
9(43%)
3(28%)
0
0
4(14%)
5(21%)
results in structures forming at the junction. Combinations consisting of
H12/12.. .F behave in the same way and do not form structures at the junction.
As can be seen from Fig. 8 (a), the value of S will fall below that of P at the
distal end of the host because S is diffusible, but the fall in value is apparently
not sufficient to initiate the formation of a new head end. In about 70 % of
12 j'12...F combinations a head forms at the junction in addition to the head
which regenerates at the distal end. This is interpreted as the concentration of
S reaching a critical value below P when the source is removed and as can be
seen from Fig. 8 (a) the value of S at the junction has fallen considerably lower
in 12/12... Fas compared with H12/12...F after 5 h. The formation of head ends
at the junction in grafts of 12/12...F, but not in H12J12...F, may be related to
Webster's finding (1966a) that a piece of tissue taken from the subhypostomal
region (region I) of an intact animal will induce a head when implanted into
the gastric region of a host animal whose own head has been removed, but is
absorbed if the host is intact.
The formation of feet at the junction in 12/12...F combinations but not in
H12J12...F may be interpreted as resulting from the greater fall in the value of
S in the graft 12 region in the former case which allows a foot end to form from
its proximal end.
In H123/12...F and H123*/12...F combinations the formation of distal
structures at the junction can again be interpreted in terms of the relationship
between S and P values at the junction (Fig. 8 a). It is of interest that H123/12
combinations result in normal animals. In terms of the model this may be
accounted for by the absence of the sink for S which is provided by the proximal
regions and so the value of S remains higher at the junction in H123\12 as
compared with HI23112.. .F(see simulation, Fig. 8a). Nearly all the H123/12.. .F
combinations passed through a stage when the junction became markedly
constricted and developed the visible characteristics of peduncle tissue.This
change was most evident around 24 h after grafting, but as can be seen from
Boundary regions in hydra
713
Table 2. Effect of wound on transmission of the positional signal
Results
Graft
(HI 2)
Host
(12...F)
No of
combinations
Wound between
regions 1 and 2
—
14
Wound between
1 and 2 regions
22
20 (90%) 0
1 (5%)*
1 (5%)*
\5
15(100%) 0
0
0
Control HI2...F
-wounded between
regions 1 and 2
A
N
HF
H
F
4 (29%) 2 (14%) 2 (14%) 6 (43%)
* Appeared to form at wound, not at junction.
the results, only about half of these combinations did in fact develop stable
proximal structures at the junction; the remainder regulated to give animals
whose appearance was quite normal and these have been classified as type N.
The other combinations also remained linear but in each case a single basal
disc formed level with the peduncle-like constriction. One animal formed a
single tentacle just below the constricted zone. After several days this tentacle
shortened and its tip became sticky, resembling in effect a tiny basal disc. It
was noted that in a few combinations consisting of HI23/12...F which looked
quite normal 48 h after grafting, additional bud formation occurred from the
midgastric region 72 h later. This may provide further evidence for the view
that the axial position of the budding region is determined in relation to the
head. Budding also occurred from the proximal end of the host 12 region in
combinations consisting of H123/12, but unlike HI 23/12...F, none of these
combinations formed structures at the junction. Regeneration of peduncle and
basal disc at the proximal end of the host 12 region occurred only very slowly,
taking about 1 week. By contrast, foot regeneration at the proximal end of
12/12 combinations took only 48 h.
Nearly all the H123*/12...F combinations which formed structures at the
junction developed outgrowths of a significant size from the main axis.
Experiments similar to those just described were also performed using another
species, Hydra attenuata, and these gave rather different results when compared
with the results using Hydra littoralis and will be reported elsewhere.
Transmission of the positional signal from the head end
The above experiments suggest that the head region may be the source of a
positional signal in hydra, at least with respect to head and foot formation. If
this is so, it is important to know how this influence is transmitted through the
animal. It is possible to utilize the difference in behaviour of H12/12...F and
12/12...F to investigate whether the transmission of such a signal would require
cell contact. An experiment was designed to answer the following question:
46-2
714
J. HICKLIN AND OTHERS
Table 3. Effect of removal of pieces of YL from H12/12...F
Amount of H removed from HI2
fragment
None (control)
One-quarter
Half to two-thirds
Three-quarters to
four-fifths
Results
-*
No. of
combinations
,
N
H
HF
F
10
10
28
18
10 (100%)
8 (80%)
17 (61%)
4 (22%)
0
0
2 (7%)
4(22%)
0
0
2 (7%)
6(33%)
0
2(20%)
7(25%)
4(22%)
would a wound between regions 1 and 2 of H12 affect the development of
H12/J2...F? As stated above, combinations of HI2/12...F form no structures
at the junction. If structures developed, therefore, in a wounded H12/J2...F,
this may indicate the manner in which such a signal from the head or foot end
may be transmitted.
HJ2/J2...F combinations were made in the usual way, and 30 min after
grafting, when the animals had relaxed again, a cut was made between regions
1 and 2 of the graft of each combination so that the axis was severed for about
half its width. Every 30 min for the next 4 h, the wound was prised open again
by means of two pins. For comparison, the procedure was repeated for another
group of H12/12...F, but in these animals the cut was made proximal to the
junction, between regions 1 and 2 of the host. To exclude the possibility that
any effect which might result from this treatment was due to trauma alone,
ungrafted animals wounded between regions 1 and 2 were used as controls.
The results (Table 2) show that in most combinations where the graft had
been wounded, new end structures were formed at the junction; in combinations
where the host had been wounded, very few animals formed new end structures.
All animals in the control series healed up normally.
Because the wound remained visible as a slight dent for at least 24 h it
was possible to observe how and where a new head or foot end formed in relation
to it. It was noted that all new ends tended to arise on the side of the axis where
the wound had been made, with no animal forming more than a single head
or peduncle and basal disc.
In terms of the present model, the most plausible explanation for the behaviour
of HJ2/12...F combinations wounded distal to the junction is that the wound
partially isolated the remainder of the animal from the source of signal, resulting in a fall in the level of 5 proximal to the wound and the formation of ends
at the junction. Since the edges of the wound came together rapidly each time
it was reopened, these results provide good evidence for the importance in
signalling of cell-to-cell contact and argue against the possibility that such a
signal is transmitted either via the coelenteron or through the extracellular
space.
715
Boundary regions in hydra
Table 4. Grafts between parts of the gastric (1234) region
Composition
Results
Graft
Host
combinations
N
H123
1
12
123
23
12
34
34
1
23...F
23...F
23...F
23...F
23...F
34... F
34...F
34
4B...F
20
25
20
32
24
20
21
19
15
20(100%)
9 (36%)
11 (55%)
15 (47%)
5 (21%)
20(100%)
6 (29%)
4 (21%)
15(100%)
H
0
16(64%)
3(15%)
17(53%)
19(79%)
0
15(71%)
15(79%)
0
F
HF
0
0
0
0
1 (5%)
5(25%)
0
0
0
0
0
0
0
0
0
0
0
0
If the above view of the mode of transmission of a signal from the head end
is correct, it is perhaps a little surprising that wounding proximal to the junction
in HI2112...F should have so little effect, but it should be noted that such a
wound will tend to increase the concentration of S at the junction (see 7/72/
12, Fig.
The effect of removing parts of the head region
If we are correct in assuming that there is a head end signal with a source,
it would be of value to find out how localized such a source may be. An experiment was therefore conducted in which varying amounts of the hypostome
and adjacent tentacles were removed at the time of combining HI2 with 12...F.
It is known that combinations consisting of 12/12...F often form structures
at the junction (Table 1).
The results of this experiment are given in Table 3. It would appear that the
smaller the amount of head region remaining, the greater the incidence of
formation of structures at the junction. As in the previous experiment, any
new ends tended to appear on the side of the axis where the operation had
been carried out.
Although this experiment confirms the idea of the head region as being the
source of a signal, it does not unfortunately resolve such questions as whether
production of signal is confined to a small group of cells located in some part
of the head, or whether the entire head region is involved in production. Nevertheless, the results do provide some indication that the source may be distributed
to some extent within the head.
These results are in general similar to those obtained for the foot end by
MacWilliams, Kafatos & Bossert (1970).
716
J. HICKLIN AND OTHERS
Table 5. Effect of starvation on 12/34...F grafts
Period of starvation (h)
Graft
donor
Host
donor
No. of
combinations
20
48
72
96
144
144
184
20
48
48
96
20
144
184
15
11
12
13
20
10
20
,
N
15
9
1
4
16
7
9
(100%)
(82%)
(8%)
(31%)
(80%)
(70%)
(45%)
A
—
•,
H
0
2(18%)
11(92%)
9(69%)
4(20%)
3(30%)
11 (55%)
Grafts between parts of the gastric region (1234)
Table 4 shows the results of a range of different axial combinations consisting
of grafts to parts of the gastric region proximal to region 1, such as 12/23...F>
12/34...F, 1/4B...F.
A general feature of these grafts is that, compared to the results of combinations listed in Table 1, the incidence of foot formation is lower. Grafts of
1/23...F, 12/23...F and 123J23...F give comparable results: in about half the
cases the distal 1 does not dominate the host region 2. An understanding of
this result probably requires a detailed study of the dynamics of S and F, but
we would argue that the 1 region does not produce 5 fast enough to prevent
head formation from the distal end of the host region 2. Support for this comes
from a comparison with 12/12...F which, as would be expected form structures
at the junction in a greater proportion of cases.
Effect of starvation prior to grafting
12134...F combinations give normal animals (Table 4), providing grafting
is performed immediately after cutting. However, if the host (34...F) is left to
regenerate for 1 h before grafting, it will 'escape' from inhibition by the
12 (Hicklin & Wolpert, in preparation). This behaviour provides a useful assay
and 12/34...F were therefore used to investigate the effect of starvation on the
behaviour of these combinations.
Groups of animals were removed from cultures and placed in separate dishes.
These animals received the same treatment as the main cultures, except that
they were not fed. At various times 12/34...F combinations were made using
these starved animals. In most series, graft and host animals had been starved
for the same length of time, but in two series the graft was taken from animals
starved for a longer period than the host. A control series of combinations
was using animals starved for only 20 h.
Table 5 shows that after 48 h of starvation before grafting 12/34... F combinations no longer gave normal animals. The structures formed were always head
Boundary regions in hydra
111
Table 6. Grafts between gastric region and peduncle
Combination
Graft
(a) H12
12
12
1
(b) 12
12
12
12
Host
56F
56F
56
6F
56F
6F
56
5
Results
combinations
N
20
12
15
10
48
18
42
20
20(100%)
7 (58%)
15(100%)
9 (90%)
28 (58%)
9 (50%)
40 (98%)
19 (95%)
H
0
5(42%)
0
1 (10%)
20(42%)
9(50%)
2 (2%)
1 (5%)
end structures. This result may be compared with the behaviour of HI 2112...F
combinations prepared using animals which had been starved for 13 days
before grafting. No structures were formed at the junction in these animals and
the presence of the head in these combinations may account for this finding.
Grafts between gastric region and peduncle
Grafts of distal gastric region, sometimes with the head included were combined with peduncle hosts with or without the basal disc at the proximal end.
The experiments are summarized in Table 6.
Since it had been found that both 12/34...F and 1/4B...F (Table 4) formed a
head only at the distal end and did not form any structures at the junction, it
first came as a considerable surprise to find that 42 % of J2/56F formed distal
structures at the junction as well as regenerating a head at the distal end. This
result was so unexpected that two detailed independent series of experiments
were carried out and are shown as (a) and (b) in Table 6. The head structures
which formed were rarely complete, consisting usually of only two or three
tentacles (Fig. 10). Furthermore, there was evidence from vital staining that the
tentacles had arisen from the proximal end of the graft, and not (as in the combinations discussed earlier) from the distal end of the host. A simulation of the
changes in S at the proximal end of the 12 region following combination with
host 56 or 56F is given in Fig. 8(6). The fall in the value of S is much greater
in 12/56F than in H12/56F or 12/56 within 5 h of grafting. The presence of a
basal disc in these combinations appears to be a very significant factor in determining whether any structures form at the junction and consistent with the
suggestion that the foot end may have special properties which causes it to
act as a sink for the signal from the head end.
It is worth remembering the possibility that the formation of tentacles at
the junction in \2/56FsLn.d 12/6F could be related to budding since the budding
region is positioned between the distal gastric region and the peduncle. A
reservation should also be made. It was observed that in most of the 1\6F and
718
J. H I C K L I N AND OTHERS
Fig. 10. Formation of tentacles at the junction of a J2J56F graft.
a few of the 12/56F combinations, signs of head regeneration from the top of
region 1 were not evident until 48 h had elapsed from the time of grafting; this
is 24 h later than usual at this level. Moreover, 24 h after grafting, the distal portion of some of these combinations had ballooned out and was found to
collapse when pricked with a pin. This inflation suggests that the animal may
have been under osmotic stress. As there was an indication in some cases that
the ectoderm had not healed properly at the distal end (complete healing being
a prerequisite for distal regeneration) it is possible that hydrostatic pressure
could in some way account for these results.
The formation of tentacles at the junction in one case of 1/6F may be explicable in terms of the detailed dynamics of S and P. The situation is analogous
to an isolated 1 maintaining its polarity.
Boundary regions in hydra
719
Table 7. Axial grafts of peduncle and foot at the foot end
No. of
combinations
No. of animals
forming basal disc
at junction
26
28
20
10(38%)
25(89%)
0
Composition
-
Graft
F65
65
6
-^~
Host
65. ..H
65... H
5...H
Fig. 11. Formation of a foot (arrow) at the junction of a HI...56/56 graft.
Axial grafts with peduncle and foot end
It has been suggested that the foot end may represent from a developmental
point of view a situation similar to that at the head end (see Wolpert et al.
1971) and thus grafts symmetrical to H12/12...F and 12/12...F were performed
at the foot end (Table 7). These consisted of H1...56/56F and HI...56/56. A
control series of the combination HI...5/6 was also performed. The control
graft formed a basal disc only at the proximal end of the region 6 and no
structures at the junction. The incidence of basal disc formation at the junction
720
J. HICKLIN AND OTHERS
Table 8
Composition
A
»
Graft
Host
2
3
H\3
56
H\56
HIS
H\6
Results
No. of
combinations
N
H
HF
R
18
9
15
16
19
12
10
4(22%)
0
13(87%)
0
0
7(58%)
2(20%)
3(17%)
2(22%)
0
0
0
0
0
0
1(11%)
0
0
3(16%)
0
0
1 (6%) 7 (39%) 3 (17%)
0
2 (22%) 4(44%)
2(13%) 0
0
16(100%)
16(84%) 0
0
5(42%) 0
8(80%) 0
12...F
12. ..F
12...F
12... F
12. ..F
12. ..F
12...F
I
was considerably higher in HI...56156 than in H1...56/56F (P<0-02) (Fig. 11).
In those grafts of HI... 56/56F which did not form a disc at the junction, regulation of peduncle length appeared to take 4 or 5 days, while the size of the basal
disc remained, as far as could be ascertained, constant.
The results of the above experiment indicate that the formation of the foot
at the proximal end may be inhibited by the presence of a grafted foot. MacWilliams & Kafatos (1968) showed that a grafted basal disc has an inhibitory
effect on the formation of another foot in its vicinity, and the above experiment
which was conducted along similar lines to theirs gave effectively the same result.
The idea that inhibition may be effected by a substance produced by the disc
itself (MacWilliams & Kafatos, 1968) is clearly tempting. This may also be interpreted in terms of a positional signal generated from the foot end in addition
to one from the head end (Wolpert, 1969; Wolpert et al. 1971).
Changes in polarity
The question of polarity changes has not entered the discussion so far because
in all the combinations described above graft and host regions were combined
with the same polarity and all grafts were such that the order of the regions was
very little altered: distal regions were at the distal end and proximal regions
at the proximal end. In no case did a change of polarity take place. We will
now consider the results of experiments in which changes in polarity did take
place, but we will first describe what happens when various symmetrical combinations are made consisting of pieces grafted with opposite polarity. Fifteen
4321/1234 combinations were performed and 11 of these regenerated a foot
at each end and formed two heads at the junction. Two heads also formed at
the junction in F...21/12...F; twelve combinations were made. Of fourteen
1234/4321, all regenerated a head at each end and a single foot at the junction.
Again only one foot formed at the junction in grafts of H...56/65...H. Ten of
these combinations were performed.
The interpretation of these results is that when symmetrical pieces are grafted
together with opposite polarity, each behaves like a separate positional field,
Boundary regions in hydra
721
Fig. 12. Development of a head, at the junction and a foot {arrow) at the distal end
of a 56112...F.
that is, it would appear that at the junction, each part of the combination is
establishing a new boundary even though the pieces have healed together. This is
what we would expect in terms of our model since the concentration, of S will
be little affected by such grafts. We are as yet unable to explain the detailed
difference in the numbers of structures forming at the junction.
The behaviour of these symmetrical grafts may be contrasted with experiments in which grafts of middle and lower gastric region or peduncle were
combined with 12...F hosts with the same polarity (Table 8). For comparison
with 3/12...F and 56/12...F, combinations consisting of H\3\12...F and
B\56\12...Frespectively were made. In all combinations consisting oi56\12...F
the polarity of the graft 56 region became reversed: a foot formed at the distal
end of the graft region 5. This is an important result since polarity reversal
occurred although the two pieces have been grafted together with the same
polarity. Twelve of these combinations formed a single head at the junction,
but in the other four animals, no head formed at the junction (Fig. 12). From
our model (Fig. 9) it is evident that the head will form at the junction since S
falls considerably below P there. Also S is lowest at the graft 5 region and might
account for foot formation there. Grafts of 2/12...F and 3/12...F also result
in a significant number of polarity reversals with the formation of a foot at the
distal end of the graft. In terms of our model we again find that the value of
S in the graft 2 and 3 regions rises and does not fall, as a result of which we
722
J. HICKLIN AND OTHERS
would predict that head formation in the graft might be inhibited. In those
combinations where polarity reversal was obtained a head (and often a number
of additional tentacles) formed at the junction (Fig. 5). In a few similar combinations, though a head was formed at the junction, the graft itself did not
form any structures at its free end.
It is in line with the theory that polarity is determined by the dynamics of
S and P that placing a head at the distal end would prevent polarity reversal
in this kind of combination. All grafts of H/56/J2...F formed a foot at the
proximal end of the graft region 6. With the exception of the few combinations
that also formed a head proximal to this foot, the gastric region of the host
(12...F) became entirely consumed by developing buds. (It was, furthermore,
interesting to find that similar budding behaviour occurred when the E\56\
12.. .F were made from animals which had been starved for 9 days before grafting,
by which time budding had in ungrafted animals entirely ceased.) Over a period
of a few days, it was noticed that some H\5\12.. .F and H16112.. .F combinations
regained the typical form of the animal, suggesting that assimilation of the
grafted peduncle had occurred. Whereas all nine 3/12...F formed structures
at the junction, only two out of 15 H/3 \12... F did so; these two animals formed
a short peduncle and basal disc at the junction which appeared to arise from
the proximal end of the graft region 3.
Another instance of a grafting arrangement which resulted in polarity reversal was region 123 combined with region 1 with the same polarity (giving
123/1). Such combinations usually produced heads at both ends and a foot at
the junction, indicating that the polarity of the graft region 1 was reversed. The
possibility should, however, be considered that the head was formed from the
proximal end of the graft region 1 because it was a free cut surface: this could
for example lead to a greater loss of S there.
When a head was grafted to the proximal end of the peduncle (i.e. H1...56/H)
it was observed that the foot did not usually form at the junction, but some way
from it. Ten out of 14 animals gave this result; in two instances only, the foot
formed at the junction; in the other two animals the peduncle seemed to
disappear entirely and budding was observed to take place midway between the
two heads where eventually a single median basal disc was formed. A comparable experiment grafting the foot to the distal end did not, however, have
a comparable effect. Fifteen grafts of F/12...F were made and in every case the
head regenerated at the distal end of the region 1 and the grafted foot was cast
off from the host, usually at the tip of an elongating tentacle.
DISCUSSION
In general, the results described here are consistent with the original suggestion of Webster (1966 a) and can be accounted for in terms of a model based on
two gradients. In the present formulation only one of the gradients, the posi-
Boundary regions in hydra
723
tional signal S is diffusible. The model is particularly successful in accounting
for the formation of boundary regions in such combinations as H12/12...F,
12/12...F and 12/56Fand H12/56F, and these have been discussed above. The
simple computer simulation of the diffusion of S shows that a semi-quantitative
consistency can be claimed. This is encouraging in view of our other studies in
which we investigated the times required by an H grafted with reverse polarity
onto the proximal regions to inhibit head formation by the 1: the results were
consistent with diffusion of a substance from the grafted head (Wolpert et aL
1972). An interesting point from the computer simulations is that when the
initial conditions involve steep gradients, the main changes take place rapidly,
within the first hour with the parameters used here. After that the changes in
concentration are relatively slow.
It seems reasonable to assume that a diffusible substance is produced at the
head end. The effect of removal of pieces of the head also indicates that its
production is distributed over the head region, but does not indicate which
cells are involved. We are currently attempting to find out. There is some
evidence for the foot end being a sink for 51 but this is not established and we
do not know the relative importance of continuous breakdown of S.
The effect of keeping a wound open is consistent with the idea that cellto-cell contact is required for signalling. It is commonly thought that cell-tocell signalling might occur via low resistance junctions (Furshpan & Potter,
1968). It is not known whether these are present in Hydra, but it is very likely
because of the presence of septate desmosome and gap junctions both of which
have been implicated as the structural basis of functional coupling. Wilby &
Webster (1970a) have suggested that the absence of signal transmission in
ionic conditions where Ca2+ was much reduced might be due to uncoupling
of the cells. Our results support the idea that cell contact is necessary for communication and the necessity to keep the wound open is in line with Wakeford's
(personal communication) observation that gap junctions can be reformed
within several hours.
An important feature of all our results is that heads or feet almost always form
where there has been a wound or a cut surface. The boundary regions only form at
the end or at the junctions between grafts. This forces one to take into account the
possibility that local wounding could play a quite important role in determining
the site of boundary regions. For example, at the wound there could a loss of
S into the medium. This would account for the behaviour of 123/1 forming
heads at the ends. However it should be emphasized that a head end can form
in the absence of a cut surface as illustrated by the formation of medial hypostomes in gastric regions treated with dithiothreitol (Hicklin, Hornbruch &
Wolpert, 1969) and in the polarity reversal experiments of Wilby & Webster
(1910b).
In its present formulation S is assumed to be a positional signal, setting
the positional value P which is not diffusible. While it can be claimed with some
724
J. HICKLIN AND OTHERS
confidence that P is a relatively stable parameter, the means whereby its value
is altered away from the boundary is far from clear. This will be considered in a
later paper.
The axial grafts described here provide a number of quite useful assays.
For example, H12/12...F never gives rise to structures at the junction whereas
12/12...Fusually does. One may therefore use this graft as an assay for the time
required for a 12 to become an HI2 (Hicklin & Wolpert, in preparation). In fact
we have used the assay in this paper to investigate the role of cell contact in the
transmission of signals and the effect of removing parts of the head. The
assay can also be used to test the effect of chemicals and irradiation by grafting
a treated H12 to a 12...F (Wolpert et ah 1971). When using such assays, care
and attention should be given to effects on cell contact. Also, as shown with
12/34...F, starvation can have quite profound effects.
The mechanism of changes in polarity will only be briefly discussed here. In
general terms, our results conform with the suggestion of Lawrence (1970;
Lawrence, Crick & Munro, 1972) that polarity is determined by the slope in
the gradient (see also Webster, 1971). The grafting of a piece where the level is
low on to a piece where the level is high may result, after diffusion, in polarity
reversal of the grafted piece. We would however suggest that this alone is
inadequate to account for polarity reversal in hydra and the dynamics and
interactions between the gradients must be taken into account. The important
question is whether one can in fact account for changes in polarity in terms of
the dynamics of gradients of the type suggested by our model or whether a
structural basis to polarity, such as active transport, is required. Wilby &
Webster (19706) and Webster (1971) on the basis of their studies on polarity
reversal have concluded that the latter is the case. A detailed analysis will be
left to a later paper since more information on the dynamics of the gradients
is required. For example, in the experiments described here it can be seen why
a head can be inhibited at the distal end in 3/12...Fand
56/12...Fcombinations:
but we need to have data on the factors determining P. How does P alter in
the situations studied by Webster, where H/321 results in reversal of polarity?
It is for this reason that the dynamics must be available for a more detailed
analysis.
Some insight into the mechanism of foot end formation has been obtained
and this appears to be analogous to that for the head end. It also seems that
proximity to the head end can inhibit foot formation: for example feet often
form the junction in 12/12...F grafts but never in H12/12...F. In the following
paper (Hicklin & Wolpert, 1973a) the dynamics of foot end formation will be
examined in more detail.
This work is supported by The Nuffield Foundation.
Boundary regions in hydra
725
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{Received 16 April 1973)