/ . Embryol. exp. Morph. Vol. 30, 3, pp. 741-752, 1973 7 4 1

Printed in Great Britain

Positional information and pattern regulation inhydra: the effect of y-radiation

By J. HICKLIN1 AND L. WOLPERT1

From the Department of Biology as Applied to Medicine,The Middlesex Hospital Medical School, London W.I

SUMMARYHydra 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. Irradiatedheads could still inhibit head formation in other regions in graft combinations. The timefor head end determination in irradiated animals appeared to be increased. It is concludedthat head end regeneration need not involve cell division.

INTRODUCTION

The possibility that cell division and cell growth take a central role in headregeneration in hydra has been put forward by Burnett (1966) who claimedthat cell division occurs mainly in a subhypostomal growth zone. The existenceof 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 hasalso shown that the tissue movements from which such a localized zone wasinferred can be accounted for by a model with cell division occurring uniformlyand tissue being lost by budding and at the two ends (Campbell, 1967b). Hisconclusions are in accord with the observations of Clarkson & Wolpert (1967),who also showed that bud elongation involved cell movement and not localizedgrowth. Nor is there evidence for Burnett's (1966) suggestion that cell divisionplays an important role in regeneration of the head. Park (1958) observed thatregeneration could still occur after doses of X-rays as high as 30000 rad; shealso found no increase in cell division during regeneration (Park, Ortmeyer &Blankenbaker, 1970). Clarkson (1969) found that inhibition of DNA synthesisin regenerating animals by hydroxyurea only slightly delayed the time requiredfor head determination. It is thus reasonable to assume that regeneration inhydra is morphollactic (Wolpert, Hicklin & Hornbruch, 1971): when the headis removed a new boundary region is established at the cut end and the newhead formed from the existing tissues.

1 Authors' address: Department of Biology as Applied to Medicine, The MiddlesexHospital 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 theelaboration of missing structures, the fact that cell division may take a criticalrole at some stage requires consideration, since several lines of evidence suggestthat cell division is a prerequisite for change in the determinative state of acell. The evidence and arguments for the role of cell division in differentiationhave been put forward by Holtzer (1971) who has developed the notion ofquantal mitosis. Gurdon & Woodland (1970) have also emphasized the roleof mitosis. It is not known whether the formation of a boundary region or thespecification of positional information is equivalent to determination or differen-tiation; however, Lawrence, Crick & Munro (1972) have suggested that changesin a gradient in the epidermal cells of the insect segment which might relateto 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 posi-tional information in hydra. The assay is to make axial grafts and then observewhether 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 thejunction whereas 12/12...F almost always does. This, and other results, havebeen interpreted in terms of two gradients. One gradient of a diffusible sub-stance, S, is the positional signal which is made at the head end; the othergradient is of positional value P. If S falls a threshold distance below P a newhead end will form. As ionizing radiation is known to block cell division wehave investigated whether y-radiation affects the behaviour of grafts; we wereinterested to see whether for example a new head end could form when no celldivision occurs.

MATERIALS AND METHODS

Hydra littoralis was used for all the experiments. The animals were grownfrom a clone and details of culture methods and selection of animals are givenin Webster & Wolpert (1966). Intact animals in groups of 12 were exposed toy-radiation from a cobalt-60 source using the procedure described in Clarkson6 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). Animalsselected for mitotic counts were fixed in Lavadowsky's fluid, and serial sections7 ju,m thick were cut perpendicular to the main axis and stained with Feulgen.Sections were examined under oil immersion with a Zeiss binocular microscopeusing a magnification of x 1000. The mitotic activity of each animal wasdetermined from counts on every tenth section, and an estimate was made ofthe mitotic index per animal. Axial grafts were performed as described inHicklin et al. (1973), using the scheme of notation for designating the differentregions 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 followingdoses of y-rays above 30000 rad (300 J/kg), and at this dose the survivalrate at 24 h was only about 10 %. The survival rate was much better after25000 rad: about 50% of irradiated animals survived 48 h or more, thoughsome degree of tentacle degeneration and disintegration of the endodermnearly always occurred. It was noticed that animals starved for 72 h beforeirradiation were more resistant to these effects. As previously noted by Park(1958), the hypostome, basal disc and budding regions were the last parts ofthe animal to disintegrate. Because the cells in animals which had been irradiatedwere in a necrotic state, micro-organisms flourished and although every attemptwas made to keep irradiated animals reasonably clean, some animals succumbedto 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 groupsof 36 animals immediately after washing. One group, A, comprised intactanimals; group B had their heads removed by cutting at the top of region 1and in group C the proximal half of the peduncle (6F) was removed. Similargroups of unirradiated animals were set up as controls. The main changeswere assessed over a 48 h period from the start of the experiment.

Twenty of the animals in the intact irradiated group (group A) were stillalive 48 h later, but the condition of the tentacles in many of these animalshad deteriorated and endoderm cells were loose in the coelenteron. Twenty-four hours from the time of irradiation, five animals were observed to possessa bud at a very early stage of development (stages 1 and 2, Clarkson & Wolpert,1967) - considerably fewer than in the unirradiated intact series where 30animals 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 basaldisc (stages 3-6), whereas stage 1 and 2 buds were visible on animals in all theunirradiated control groups. Tentacle morphogenesis appeared to take slightlylonger in buds on irradiated animals as compared with controls. In their analysisof the main changes in form occurring in budding, Clarkson & Wolpert (1967)have indicated that the main period of bud outgrowth from the parent axisoccurs during the first 9 h from the time the bud is just visible as a small conicalprotuberance (stage 1 to stage 3) and that a further 30 h is required for morpho-genesis of tentacles and basal disc. The discovery of stage 1 and stage 2 buds onsome irradiated animals 24 h after their exposure to the source suggests thatbud initiation can still occur after such a high dose of y-rays. The possibilityshould, however, be considered that these buds may have already been deter-mined before the parent animals were irradiated. Moreover, although Clarkson

48 E M B 30

744 J. HICKLIN AND L. WOLPERT

Fig. 1. A hydra which received 25000 rad and then had hypostome and tentaclesexcised; 72 h later it has fully regenerated at the head end.

& Wolpert (1967) showed that elongation of the bud from stage 1 to stage 2was unaffected by 25000 rad, it is not known whether tentacle morphogenesisbegan 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 inthese the regeneration of missing distal structures appeared to be at about thesame stage as in the control, i.e. hypostome and short tentacles, but in theirradiated group the regenerating tentacles appeared to be slightly shorterthan those of the control. This difference was very much more marked aftera further 24 h, by which time all control animals had regenerated fully, butthe 24 surviving irradiated animals had tentacles which were only about aquarter 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 tohave completely regenerated (Fig. 1), indicating that in these animals tentacleelongation may have been slowed down. However, this apparent slow develop-

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746 J. HICKLIN AND L. WOLPERT

Table 1. Effect of a dose of 25000 rad on the mitotic activity in intact andregenerating hydra

Time offixation post-irradiation

(h)

4

24

48

Samples

Controls

Irradiatedanimals

Controls

Irradiatedanimals

Controls

Irradiatedanimals

Total cellscounted

30355765

37544360

17742470

1714216239294041

14041524

Intact hydra

Total normalmitoses

2836

00

1329

00

1730

00

^Mitoticindex

0-92%0-63%

00

0-73%1-17%

00043%0-74%

00

Regeneratingi

Total cells 'counted

48543746

37212276

26452969

1276102421151827

830971

hydra

Fotal normal Mitoticmitoses

7169

00

4352

00

199

01

index

1-46%1-84%

00

1-63%1-75%

00090%0-50%

00

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 regenerationbetween the behaviour of the irradiated and control groups. Foot regenerationwas assessed by testing for the adhesive properties of the basal disc on a cleanglass surface (Hicklin & Wolpert, 1973). A few animals in both group C andits control had regenerated a basal disc by 24 h after cutting, and all after 48 hof regeneration.

Effect of radiation on cell division

In order to determine the effect of radiation on the mitotic activity of intactand regenerating animals, animals were exposed to 25000 rad as describedabove. Intact animals and animals regenerating the head were fixed 4, 24 and48 h after irradiation and prepared for histological examination. Unirradiatedcontrols for both these groups were also run. Two animals from each group werecounted.

No normal mitotic figures were seen in any of the sections examined from theirradiated animals. Among the cells counted from animals of both 4 h serieswere a small number (between six and eight per animal) with pycnotic nuclei(Fig. 2 A). Evidence of chromosome damage was further shown by the forma-tion of 'bridges' between the separating chromosomes in a single anaphasestage 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

H12*H12H12*Control H12H12*H12H12*HI I*Control / / / /

of host

12...F12*...F12*...F12. ..F34...F34*...F34*. ..F23. ..F23...F

N = normal animal; H = headat junction.

* Irradiated regions.

combinations

141513101412151415

at junction; HF

N

5 (36%)15(100%)11 (85%)10(100%)12 (88%)14(100%)14 (94%)14(100%)15(100%)

H

000000000

HF

2 04%)00000000

F

7(50%)0

2(15%)0

2(12%)0

1 (6%)00

= head and foot at junction; F = foot

of any mitotic activity in the animals of either 24 h and 48 h series: one or twocells were noted per animal count with fused and adherent chromosomes, andonce (in an animal which had been regenerating 48 h) a metaphase stage inwhich fragmented chromosomes were visible. Apart from the absence of normalmitoses the histology of the irradiated animals appeared to be little differentfrom that of the unirradiated controls. There was, however, a marked absenceof nests of I-cells in irradiated animals 24 h and 48 h after treatment, which isconsistent with the apparent inhibition of mitotic activity in these animals.The total number of cells counted per animal, together with the total numberof 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 inall] four major cell types. It is clear that in the irradiated animals normalmitosis had been completely suppressed. The overall mitotic index was higherin unirradiated regenerating animals compared with intact controls, but as theanimals were fixed in two different occasions this most likely represents differ-ences in nutritional state (Campbell, 1967a).

Effect of irradiation on positional information

Since our results indicated that a high dose of y-irradiation virtuallysuppresses cell division and yet allowed regeneration to occur, it is of interestto determine whether the production, transmission or response to a positionalsignal may in any way have been interfered with in animals which have beenirradiated and in which cell division has ceased. The grafting experimentswhich 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 moreproximal regions in the absence of the head

Compositionof graft

12*12Control 1212*1212*Control 12

Compositionof host

12 .12*.12 .34 .34*.34*.34 .

..F

..F

..F

..F

..F

..F

..F

No. ofcombinations

15121513151012

* Irradiated

r

293

12149

12

N

(13%)(75%)(20%)(92%)(94%)(90%)

(100%)regions.

232111

Results

H

(13%)(25%)(13%)(8%)(6%)

(10%)0

HF

9(61%)0

7(47%)0000

F

2(13%)0

3 (20%)0000

or the host or both had been obtained from irradiated animals (see Table 2).Control combinations were made with unirradiated hosts and grafts; a seriesof combinations consisting of irradiated H12 on to unirradiated 34...F wasalso made. Only those combinations which appeared to be in a reasonablestate of health (i.e. no significant loosening of endoderm or sloughing of cellsat the junction) were included in the results. These were assessed 24 h and 48 hafter grafting.

As can be seen from Table 2 the structures formed at the junction mainlywhen the H12 only was irradiated. Where structures did form at the junction,these were usually foot end structures (consisting of a peduncle and basaldisc), but in two of the irradiated H12 grafts onto unirradiated 12...F hosts, asingle tentacle was formed immediately below the proximal axis at the junction.Irradiated H12/12...Fcombinations healed together badly, presumably becausethe cells were in a necrotic state, and in some cases structures formed at thejunction. These results suggest that the production of the signal is being atten-uated and this is supported by the fact that irradiated HI 2 on 34...Fare nearlyall normal.

Fewer structures formed at the junction when both graft and host had beenirradiated, but budding always continued in irradiated hosts and, just as inunirradiated controls, new buds were initiated after 48 h from the time of graft-ing. It was noted occasionally that the last bud to be formed in irradiated H12onto unirradiated 12...Ffailed to detach. This could be linked with the dissolu-tion of the distal part of the combination as a result of radiation damage.

Further evidence indicating that signalling may not be affected by high dosesof radiation comes from combinations in which only a central section wasirradiated, that is H\1\23...F. In these, transmission of signal again did notappear 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 ofgrafting H12 on to irradiated regenerating 12...F

Peiiod of Resultsregeneration No. of , A *

State of 12...F (h) combinations N H

Control 6 13 0 13(100%)Irradiated 6 14 4(29%) 10 (71%)Control 8 10 0 10(100%)Irradiated 8 10 5(50%) 5 (50%)

removed. From previous work (Hicklin et ah 1973) it is known that 72/72...Fusually 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 grafted72 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 graftsof this type. When either or both parts of 12/34...F combinations had beenobtained from donor animals which had previously been irradiated, normalanimals resulted in almost every case, and in this way behaved just like controls.Irradiated 72 onto unirradiated 72...Fhost very often formed structures at thejunction, but in all cases there was evidence of head regeneration from theregion 7 of the graft within 48 h and in none of the animals was a foot observedto form there, i.e. no instances of polarity reversal. It can be seen that the pro-portion of combinations forming structures at the junction was much lower whenan 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 togive normal animals (Wolpert et al. 1971; Hicklin et al. 1973). If, however, the72...F host is allowed to regenerate for about 5 h before being combined withthe 7/72 graft, a head will form at the junction; this indicates that a new headboundary region has been determined at the distal end of the host region 7.The following experiment was performed along the same lines to investigatewhether the time at which head end determination takes place in regeneratinganimals is affected by such high doses of radiation. It has already been shownabove that HI2/12...F are normal.

Animals were irradiated as before, and the head was removed immediatelyafter washing. After 6 h the distal end which had healed was re-opened, and a7/72 region was grafted on to it from an intact animal which had not beenirradiated. It was found in some cases that the H12 did not always readily com-bine 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. Controlseries were set up using unirradiated animals. The results are shown in Table 4.

750 J. HICKLIN AND L. WOLPERT

Fig. 3. Formation of head end (arrow) at the junction between an unirradiatedHI 2 region and an irradiated 12...F host which had been allowed to regenerate6 h before grafting. Photograph taken 48 h after grafting.

All the control combinations formed heads at the junction: a high proportionof combinations with irradiated hosts also did so (Fig. 3). The results show thathead end determination occurs in irradiated regenerating animals, but the timerequired is longer.

DISCUSSION

High doses of y-irradiation (25000 rad) seem to have remarkably little effecton pattern regulation and regeneration in hydra as judged by both normal re-generation and various grafting assays. Irradiation seems to have some effecton the production of an inhibitory influence at the head end, but the ability totransmit the inhibitory signal seems relatively unaffected. In addition, the abilityto form a new head or foot and the time for head formation seem relativelynormal. 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 sup-press normal cell division as judged by the absence of normal mitotic figures.This would suggest that cell division is not important for regeneration or patternregulation (Wolpert et al. 1971) but may be required for new bud initiation(Webster & Hamilton, 1972). While at first sight one might think that ourresults are at variance with those suggestions that link cell division to deter-mination and differentiation, it is important to realize that our assay is normalmitosis. Thus other phases of the cell cycle such as nuclear membrane break-down could still be crucial features of determination or differentiation. Also,our results refer only to changes at the boundary region and not to changeselsewhere or requirements for reversal of polarity. It is thus of great interestthat Cooke (1973) has evidence in amphibian embryos that the setting up ofnew positional fields and the cells' interpretation of these fields in terms ofnormal cellular differentiation, can occur even when cell division is blocked.

Our results with irradiation may be contrasted with those obtained withcertain chemical agents such as dithiothreitol (Hicklin, Hornbruch & Wolpert,1968) and colchicine (Corff & Burnett, 1969; Wolpert et al. 1971), where treat-ment of distal regions results in the formation of feet at the boundary and inreversal of polarity. This has never been observed with irradiated animals.

This work is supported by The Nuffield Foundation.

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LAWRENCE, P. A., CRICK, F. H. C. & MUNRO, M. (1972). A gradient of positional informa-tion in an insect, Rhodnius. J. Cell Sci. 11, 815-854.

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(Received 16 April 1973, revised 10 June 1973)