/ . Embryol. exp. Morph. Vol. 30, 3, pp. 741-752, 1973
Printed in Great Britain
741
Positional information and pattern regulation in
hydra: the effect of y-radiation
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
Hydra exposed to high doses of y-irradiation (25000 rad) are still capable of regeneration
although no normal mitotic figures could be seen for up to 48 h after irradiation. Irradiated
heads could still inhibit head formation in other regions in graft combinations. The time
for head end determination in irradiated animals appeared to be increased. It is concluded
that head end regeneration need not involve cell division.
INTRODUCTION
The possibility that cell division and cell growth take a central role in head
regeneration in hydra has been put forward by Burnett (1966) who claimed
that cell division occurs mainly in a subhypostomal growth zone. The existence
of such a localized growth zone has not been demonstrated and Campbell's
(1967<z) careful study has shown that growth in hydra is not localized. He has
also shown that the tissue movements from which such a localized zone was
inferred can be accounted for by a model with cell division occurring uniformly
and tissue being lost by budding and at the two ends (Campbell, 1967b). His
conclusions are in accord with the observations of Clarkson & Wolpert (1967),
who also showed that bud elongation involved cell movement and not localized
growth. Nor is there evidence for Burnett's (1966) suggestion that cell division
plays an important role in regeneration of the head. Park (1958) observed that
regeneration could still occur after doses of X-rays as high as 30000 rad; she
also found no increase in cell division during regeneration (Park, Ortmeyer &
Blankenbaker, 1970). Clarkson (1969) found that inhibition of DNA synthesis
in regenerating animals by hydroxyurea only slightly delayed the time required
for head determination. It is thus reasonable to assume that regeneration in
hydra is morphollactic (Wolpert, Hicklin & Hornbruch, 1971): when the head
is removed a new boundary region is established at the cut end and the new
head formed from the existing tissues.
1
Authors' address: Department of Biology as Applied to Medicine, The Middlesex
Hospital Medical School, London W1P 6DB, U.K.
742
J. HICKLIN AND L. WOLPERT
Even though cell division does not appear to be a significant factor in the
elaboration of missing structures, the fact that cell division may take a critical
role at some stage requires consideration, since several lines of evidence suggest
that cell division is a prerequisite for change in the determinative state of a
cell. The evidence and arguments for the role of cell division in differentiation
have been put forward by Holtzer (1971) who has developed the notion of
quantal mitosis. Gurdon & Woodland (1970) have also emphasized the role
of mitosis. It is not known whether the formation of a boundary region or the
specification of positional information is equivalent to determination or differentiation; however, Lawrence, Crick & Munro (1972) have suggested that changes
in a gradient in the epidermal cells of the insect segment which might relate
to positional information only occur when the cells divide.
In previous papers (Wolpert et al. 1971; Hicklin, Hornbruch, Wolpert &
Clarke, 1973) models and assays have been developed for investigating positional information in hydra. The assay is to make axial grafts and then observe
whether head or feet develop at the junctions and at the ends. For example,
H12/12...F (for terminology see earlier papers) never gives heads or feet at the
junction whereas 12/12...F almost always does. This, and other results, have
been interpreted in terms of two gradients. One gradient of a diffusible substance, S, is the positional signal which is made at the head end; the other
gradient is of positional value P. If S falls a threshold distance below P a new
head end will form. As ionizing radiation is known to block cell division we
have investigated whether y-radiation affects the behaviour of grafts; we were
interested to see whether for example a new head end could form when no cell
division occurs.
MATERIALS AND METHODS
Hydra littoralis was used for all the experiments. The animals were grown
from a clone and details of culture methods and selection of animals are given
in Webster & Wolpert (1966). Intact animals in groups of 12 were exposed to
y-radiation from a cobalt-60 source using the procedure described in Clarkson
6 Wolpert (1967). The dose rate from the source was 5-3 kR/min (1-3 C/kg/min)
as measured by ferrous sulphate dosimetry (Miller & Wilkinson, 1952). Animals
selected for mitotic counts were fixed in Lavadowsky's fluid, and serial sections
7 ju,m thick were cut perpendicular to the main axis and stained with Feulgen.
Sections were examined under oil immersion with a Zeiss binocular microscope
using a magnification of x 1000. The mitotic activity of each animal was
determined from counts on every tenth section, and an estimate was made of
the mitotic index per animal. Axial grafts were performed as described in
Hicklin et al. (1973), using the scheme of notation for designating the different
regions of hydra and the method for scoring the results given there.
The effect of y-radiation on hydra
743
General effects on animals of high doses of y-rays
Very few irradiated animals were able to survive for more than 24 h following
doses of y-rays above 30000 rad (300 J/kg), and at this dose the survival
rate at 24 h was only about 10 %. The survival rate was much better after
25000 rad: about 50% of irradiated animals survived 48 h or more, though
some degree of tentacle degeneration and disintegration of the endoderm
nearly always occurred. It was noticed that animals starved for 72 h before
irradiation were more resistant to these effects. As previously noted by Park
(1958), the hypostome, basal disc and budding regions were the last parts of
the animal to disintegrate. Because the cells in animals which had been irradiated
were in a necrotic state, micro-organisms flourished and although every attempt
was made to keep irradiated animals reasonably clean, some animals succumbed
to overwhelming infections. It was noted that cutting aggravated the process.
In the experiments which follow, the dose used was 25000 rad.
Effect of radiation on budding and on head and foot regeneration
Nine batches of intact animals were irradiated and divided into three groups
of 36 animals immediately after washing. One group, A, comprised intact
animals; group B had their heads removed by cutting at the top of region 1
and in group C the proximal half of the peduncle (6F) was removed. Similar
groups of unirradiated animals were set up as controls. The main changes
were assessed over a 48 h period from the start of the experiment.
Twenty of the animals in the intact irradiated group (group A) were still
alive 48 h later, but the condition of the tentacles in many of these animals
had deteriorated and endoderm cells were loose in the coelenteron. Twentyfour hours from the time of irradiation, five animals were observed to possess
a bud at a very early stage of development (stages 1 and 2, Clarkson & Wolpert,
1967) - considerably fewer than in the unirradiated intact series where 30
animals carried very young buds. Buds on irradiated animals (groups A, B, C)
after 48 h were all at later stages, i.e. long buds to buds with their own basal
disc (stages 3-6), whereas stage 1 and 2 buds were visible on animals in all the
unirradiated control groups. Tentacle morphogenesis appeared to take slightly
longer in buds on irradiated animals as compared with controls. In their analysis
of the main changes in form occurring in budding, Clarkson & Wolpert (1967)
have indicated that the main period of bud outgrowth from the parent axis
occurs during the first 9 h from the time the bud is just visible as a small conical
protuberance (stage 1 to stage 3) and that a further 30 h is required for morphogenesis of tentacles and basal disc. The discovery of stage 1 and stage 2 buds on
some irradiated animals 24 h after their exposure to the source suggests that
bud initiation can still occur after such a high dose of y-rays. The possibility
should, however, be considered that these buds may have already been determined before the parent animals were irradiated. Moreover, although Clarkson
48
E M B 30
744
J. H I C K L I N AND L. W O L P E R T
Fig. 1. A hydra which received 25000 rad and then had hypostome and tentacles
excised; 72 h later it has fully regenerated at the head end.
& Wolpert (1967) showed that elongation of the bud from stage 1 to stage 2
was unaffected by 25000 rad, it is not known whether tentacle morphogenesis
began at the same time in buds from irradiated parents and unirradiated controls.
Twenty-eight of the animals in group B were still alive 24 h later, and in
these the regeneration of missing distal structures appeared to be at about the
same stage as in the control, i.e. hypostome and short tentacles, but in the
irradiated group the regenerating tentacles appeared to be slightly shorter
than those of the control. This difference was very much more marked after
a further 24 h, by which time all control animals had regenerated fully, but
the 24 surviving irradiated animals had tentacles which were only about a
quarter to a third of the length of those of the control regenerated animals.
Only after 72 h of regeneration did any of the irradiated animals appear to
have completely regenerated (Fig. 1), indicating that in these animals tentacle
elongation may have been slowed down. However, this apparent slow develop-
Fig. 2. Irradiated animal receiving 25000 rad and fixed 4 h after treatment. (A) Pycnotic nuclei in two adjacent cells of ectoderm (arrows). (B) Abnormal anaphase in another ectoderm cell; chromosome 'stickiness' resulting in bridge-formation between
separating chromosomes. Arrow indicates position of bridges. Phase, x 1000.
A
t
vv va*
r
o
a"
•s,
746
J. HICKLIN AND L. WOLPERT
Table 1. Effect of a dose of 25000 rad on the mitotic activity in intact and
regenerating hydra
Time of
fixation postirradiation
Samples
(h)
4
Controls
Irradiated
animals
24
Controls
Irradiated
animals
48
Controls
Irradiated
animals
Intact hydra
Regenerating hydra
i
Total cells Total normal
counted
mitoses
Mitotic^ Total cells 'Fotal normal
counted
index
mitoses
Mitotic
index
3035
5765
28
36
0-92%
0-63%
4854
3746
71
69
1-46%
1-84%
3754
4360
1774
2470
0
0
13
29
0
0
0-73%
1-17%
3721
2276
2645
2969
0
0
43
52
0
0
1-63%
1-75%
1714
2162
3929
4041
0
0
17
30
0
0
043%
0-74%
1276
1024
2115
1827
0
0
19
9
0
0
090%
0-50%
1404
1524
0
0
830
971
0
1
0
0
0
0
ment may, in fact, have been due to attrition or absorption of the tentacles.
These results are similar to those obtained by Bhattach (1971).
In group C no significant differences were observed in foot regeneration
between the behaviour of the irradiated and control groups. Foot regeneration
was assessed by testing for the adhesive properties of the basal disc on a clean
glass surface (Hicklin & Wolpert, 1973). A few animals in both group C and
its control had regenerated a basal disc by 24 h after cutting, and all after 48 h
of regeneration.
Effect of radiation on cell division
In order to determine the effect of radiation on the mitotic activity of intact
and regenerating animals, animals were exposed to 25000 rad as described
above. Intact animals and animals regenerating the head were fixed 4, 24 and
48 h after irradiation and prepared for histological examination. Unirradiated
controls for both these groups were also run. Two animals from each group were
counted.
No normal mitotic figures were seen in any of the sections examined from the
irradiated animals. Among the cells counted from animals of both 4 h series
were a small number (between six and eight per animal) with pycnotic nuclei
(Fig. 2 A). Evidence of chromosome damage was further shown by the formation of 'bridges' between the separating chromosomes in a single anaphase
stage which was noted in one animal (Fig. 2B). There was virtually no indication
The effect of y-radiation on hydra
141
Table 2. The effect of irradiation on axial grafts between distal regions,
including the head, and more proximal regions
Results
of graft
of host
combinations
N
H
HF
F
H12*
14
5 (36%) 0
12...F
2 04%) 7(50%)
H12
0
0
15
12*...F
15(100%) 0
H12*
0
13
12*...F
11 (85%) 0
2(15%)
0
10
12. ..F
0
Control H12
10(100%) 0
14
0
H12*
34...F
12 (88%) 0
2(12%)
12
0
0
H12
34*...F
14(100%) 0
0
15
H12*
34*. ..F
14 (94%) 0
1 (6%)
0
14
HI I*
0
23. ..F
14(100%) 0
0
15
0
23...F
Control / / / /
15(100%) 0
N = normal animal; H = head at junction; HF = head and foot at junction; F = foot
at junction.
* Irradiated regions.
of any mitotic activity in the animals of either 24 h and 48 h series: one or two
cells were noted per animal count with fused and adherent chromosomes, and
once (in an animal which had been regenerating 48 h) a metaphase stage in
which fragmented chromosomes were visible. Apart from the absence of normal
mitoses the histology of the irradiated animals appeared to be little different
from that of the unirradiated controls. There was, however, a marked absence
of nests of I-cells in irradiated animals 24 h and 48 h after treatment, which is
consistent with the apparent inhibition of mitotic activity in these animals.
The total number of cells counted per animal, together with the total number
of these cells showing normal mitotic figures, and the calculated mitotic index,
are given in Table 1. Mitotic figures were observed throughout the axis
(except in the tentacles and basal disc) in all the unirradiated animals and in
all] four major cell types. It is clear that in the irradiated animals normal
mitosis had been completely suppressed. The overall mitotic index was higher
in unirradiated regenerating animals compared with intact controls, but as the
animals were fixed in two different occasions this most likely represents differences in nutritional state (Campbell, 1967a).
Effect of irradiation on positional information
Since our results indicated that a high dose of y-irradiation virtually
suppresses cell division and yet allowed regeneration to occur, it is of interest
to determine whether the production, transmission or response to a positional
signal may in any way have been interfered with in animals which have been
irradiated and in which cell division has ceased. The grafting experiments
which are described below were therefore undertaken (Tables 2, 3).
Combinations were made consisting of H12jl2...F'm which either the graft
748
J. HICKLIN AND L. WOLPERT
Table 3. The effect of irradiation on axial grafts between distal and more
proximal regions in the absence of the head
Results
Composition
of graft
12*
12
Control 12
12*
12
12*
Control 12
Composition
of host
No. of
combinations
12 . ..F
12*...F
12 . ..F
34 . ..F
34*...F
34*...F
34 . ..F
15
12
15
13
15
10
12
r
N
H
2 (13%) 2 (13%)
9 (75%) 3 (25%)
3 (20%) 2 (13%)
12 (92%) 1 (8%)
14 (94%) 1 (6%)
9 (90%) 1 (10%)
0
12 (100%)
HF
9(61%)
F
2(13%)
0
0
7(47%)
3 (20%)
0
0
0
0
0
0
0
0
* Irradiated regions.
or the host or both had been obtained from irradiated animals (see Table 2).
Control combinations were made with unirradiated hosts and grafts; a series
of combinations consisting of irradiated H12 on to unirradiated 34...F was
also made. Only those combinations which appeared to be in a reasonable
state of health (i.e. no significant loosening of endoderm or sloughing of cells
at the junction) were included in the results. These were assessed 24 h and 48 h
after grafting.
As can be seen from Table 2 the structures formed at the junction mainly
when the H12 only was irradiated. Where structures did form at the junction,
these were usually foot end structures (consisting of a peduncle and basal
disc), but in two of the irradiated H12 grafts onto unirradiated 12...F hosts, a
single tentacle was formed immediately below the proximal axis at the junction.
Irradiated H12/12...Fcombinations healed together badly, presumably because
the cells were in a necrotic state, and in some cases structures formed at the
junction. These results suggest that the production of the signal is being attenuated and this is supported by the fact that irradiated HI 2 on 34...Fare nearly
all normal.
Fewer structures formed at the junction when both graft and host had been
irradiated, but budding always continued in irradiated hosts and, just as in
unirradiated controls, new buds were initiated after 48 h from the time of grafting. It was noted occasionally that the last bud to be formed in irradiated H12
onto unirradiated 12...Ffailed to detach. This could be linked with the dissolution of the distal part of the combination as a result of radiation damage.
Further evidence indicating that signalling may not be affected by high doses
of radiation comes from combinations in which only a central section was
irradiated, that is H\1\23...F. In these, transmission of signal again did not
appear to be affected since, like controls, no structures formed at the junction.
The above axial grafts were all carried out with an intact H at the distal end.
A further series of experiments were carried out in which the distal head was
The effect of y-radiation on hydra
749
Table 4. Head determination following irradiation usitig the assay of
grafting H12 on to irradiated regenerating 12...F
State of 12...F
Control
Irradiated
Control
Irradiated
Peiiod of
regeneration
(h)
6
6
8
8
Results
No. of
combinations
13
14
10
10
A
,
*
N
H
0
4(29%)
0
5(50%)
13(100%)
10 (71%)
10(100%)
5 (50%)
removed. From previous work (Hicklin et ah 1973) it is known that 72/72...F
usually gives heads and feet at the junction and always a head at the distal end,
and that 12/34...Fis always normal. It is also known that treating the grafted
72 in 72/72...F with a variety of chemicals can lead to a reversal in its polarity.
As can be seen from Table 3, y-irradiation has in general little effect on grafts
of this type. When either or both parts of 12/34...F combinations had been
obtained from donor animals which had previously been irradiated, normal
animals resulted in almost every case, and in this way behaved just like controls.
Irradiated 72 onto unirradiated 72...Fhost very often formed structures at the
junction, but in all cases there was evidence of head regeneration from the
region 7 of the graft within 48 h and in none of the animals was a foot observed
to form there, i.e. no instances of polarity reversal. It can be seen that the proportion of combinations forming structures at the junction was much lower when
an unirradiated region 72 was combined with irradiated 72...F host.
Effect of radiation on head end determination
Grafts of 7/7 2/72...T7 do not form structures at the junction, but regulate to
give normal animals (Wolpert et al. 1971; Hicklin et al. 1973). If, however, the
72...F host is allowed to regenerate for about 5 h before being combined with
the 7/72 graft, a head will form at the junction; this indicates that a new head
boundary region has been determined at the distal end of the host region 7.
The following experiment was performed along the same lines to investigate
whether the time at which head end determination takes place in regenerating
animals is affected by such high doses of radiation. It has already been shown
above that HI2/12...F are normal.
Animals were irradiated as before, and the head was removed immediately
after washing. After 6 h the distal end which had healed was re-opened, and a
7/72 region was grafted on to it from an intact animal which had not been
irradiated. It was found in some cases that the H12 did not always readily combine with the irradiated 72...F and that cells would slough off at the junction.
The procedure was repeated with other regenerating animals after 8 h. Control
series were set up using unirradiated animals. The results are shown in Table 4.
750
J. H I C K L I N AND L. W O L P E R T
Fig. 3. Formation of head end (arrow) at the junction between an unirradiated
HI 2 region and an irradiated 12...F host which had been allowed to regenerate
6 h before grafting. Photograph taken 48 h after grafting.
All the control combinations formed heads at the junction: a high proportion
of combinations with irradiated hosts also did so (Fig. 3). The results show that
head end determination occurs in irradiated regenerating animals, but the time
required is longer.
DISCUSSION
High doses of y-irradiation (25000 rad) seem to have remarkably little effect
on pattern regulation and regeneration in hydra as judged by both normal regeneration and various grafting assays. Irradiation seems to have some effect
on the production of an inhibitory influence at the head end, but the ability to
transmit the inhibitory signal seems relatively unaffected. In addition, the ability
to form a new head or foot and the time for head formation seem relatively
normal. Only the ability to initiate new buds appears to be severely impaired.
The effect of y-radiation on hydra
751
The doses of y-irradiation employed have here been shown to completely suppress normal cell division as judged by the absence of normal mitotic figures.
This would suggest that cell division is not important for regeneration or pattern
regulation (Wolpert et al. 1971) but may be required for new bud initiation
(Webster & Hamilton, 1972). While at first sight one might think that our
results are at variance with those suggestions that link cell division to determination and differentiation, it is important to realize that our assay is normal
mitosis. Thus other phases of the cell cycle such as nuclear membrane breakdown could still be crucial features of determination or differentiation. Also,
our results refer only to changes at the boundary region and not to changes
elsewhere or requirements for reversal of polarity. It is thus of great interest
that Cooke (1973) has evidence in amphibian embryos that the setting up of
new positional fields and the cells' interpretation of these fields in terms of
normal cellular differentiation, can occur even when cell division is blocked.
Our results with irradiation may be contrasted with those obtained with
certain chemical agents such as dithiothreitol (Hicklin, Hornbruch & Wolpert,
1968) and colchicine (Corff & Burnett, 1969; Wolpert et al. 1971), where treatment of distal regions results in the formation of feet at the boundary and in
reversal of polarity. This has never been observed with irradiated animals.
This work is supported by The Nuffield Foundation.
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