/ . Embryol. exp. Morph. Vol. 18, 2, pp. 181-97, October 1967 181With 1 plate

Printed in Great Britain

Studies on pattern regulation in Hydra

IV. The effect of colcemide and puromycinon polarity and regulation

By GERALD WEBSTER1

From the Zoology Department, King's College, University of London

INTRODUCTION

Regulation in hydra is polarized, the new hypostome always forming at thedistal end of an isolated piece. Polarity is rigorously maintained in such piecesbut can be altered in graft combination (Peebles, 1900; King, 1901; Morgan,1901; Browne, 1909; Goetsch, 1929; Tardent, 1960; Webster, 19666).

Previous investigations (Webster, 1966a, b; Webster & Wolpert, 1966) havesuggested that polarized regulation occurs as a result of the interaction of threefactors: time for hypostome determination, inhibition of hypostome determi-nation and threshold for inhibition. All these factors appear to be graded alongthe axis. There is some evidence that time for hypostome determination andthreshold for inhibition are closely linked factors (Webster, 19666) and inmany experiments it is not possible to distinguish between them. For thisreason they are referred to throughout this paper as 'time-threshold' factorsor properties.

If polarized regulation does result from interaction between the abovefactors, then experimental alteration of polarity would be expected to dependupon interference with these factors or with their interaction. Experimentalalteration of polarity in graft combinations has previously been discussed in suchterms (Webster, 19666) but no attention has been devoted to experimentalalteration by other means. This is the subject of the present paper.

A large body of literature exists on the effect of various environmentalfactors (chemical agents, temperature, electric current, etc.) on regulation andpolarity (see e.g. Child, 1941). In some hydroids, but not hydra, polarity isnormally rather labile and can be influenced by a variety of environmentalfactors. Tubularia is a good example (literature reviewed by Child, 1941;Tardent, 1960, 1963). Corymorpha is a hydroid which often shows bipolarregulation in short pieces and the frequency of such forms can be increased byexposing the pieces to inhibitory agents (cyanide, alcohol, low temperature, etc.)

1 Author's address: School of Biological Sciences, University of Sussex, Falmer, Brighton,Sussex, England.

12 JEEM l8

182 G. WEBSTER

for a short time after isolation. In the majority of such experiments there hasbeen no detailed analysis of the effect of such agents at either a biologicalor a biochemical level. It has usually been assumed that the alteration of polarityhas been produced as a result of interference with some sort of axial gradientwhich 'controls' polarity but little convincing evidence has been advanced tosupport this view.

It is surprising that little work along these lines has been carried out withhydra. Lesh & Burnett (1964,1966) have recently demonstrated that a substanceis present in homogenates of H. pirardi and H. viridis which when applied toisolated pieces of H. pirardi is capable of altering polarity. Lentz (1965), usingslightly different techniques, has made a similar observation. All these workershave interpreted their results in terms of Burnett's (1961) hypothesis of controlof distal regeneration by' growth stimulating' and' growth inhibiting' substances.

The basis for the experiments described in this paper is the work of Flickinger(Flickinger, 1959; Flickinger & Coward, 1962), originating from the observa-tions of Kanatani (1958) that brief treatment of planaria with colcemide(desacetylmethylcolchicine) causes bipolar regulation in isolated pieces. Flick-inger showed that there was a gradient in the rate of incorporation of labelledamino acids into protein along the body of the animal, and demonstrated thattreatment with colcemide or chloramphenicol flattened or abolished thisgradient. Thus the morphological effect of colcemide could be related to aspecific biochemical effect.

In this paper experiments are described on the effect of colcemide on polarizedregulation in various isolated regions of hydra. The effect of this substance onhypostome formation and the properties of the determined and 'adult' hypo-stome are also considered. Experiments have also been carried out to determinethe effect of colcemide on those properties (' time-threshold' properties) whichare believed to control polarity. Similar experiments have been carried outusing puromycin, also an inhibitor of protein synthesis (Yarmolinsky &de la Haba, 1959), since preliminary experiments indicated that chloramphenicol—effective on planaria—is very toxic to hydra.

It must be emphasized that the primary purpose of the experiments was toinvestigate how these chemical agents affect polarity at a biological level, andto consider the results in terms of concepts of control which have been developedto explain normal regulation. No attempt is made to discuss the possiblebiochemical effects of these substances and no assumptions are made thatthey are acting in any particular way at a biochemical level. Such considerationmust await the completion of biochemical investigations which are still inprogress.

Effect of colcemide on Hydra polarity 183

MATERIALS AND METHODS

Hydra littoralis were used for all experiments. Details with regard to selectionof animals, treatment during experiments, etc., have been given in Webster &Wolpert (1966).

Colcemide (a gift from Ciba Laboratories, Horsham, Sussex) and puromycindihydrochloride (Nutritional Biochemicals Corp.) were dissolved in ' M 'solution and animals were treated in large volumes of the solution at 26 °C.After treatment they were washed three times in ' M \

Different regions of the axis were isolated as follows: digestive zones bycuts just proximal to the ring of tentacles and just distal to the budding zone;peduncles by a cut just proximal to the budding zone. Hypostomes and tentaclesor peduncles were removed as in the isolation of the digestive zone. Animalswere allowed to extend maximally before cutting.

Transplantation experiments to investigate hypostomal or subhypostomalproperties were performed as previously described (Webster & Wolpert, 1966;Webster, 1966 a, b).

EXPERIMENTS AND RESULTS

General effects of colcemide and puromycin

Colcemide (5 x 10~5 M, 20 /«g/ml) inhibits reconstitution of all missing parts.Hydra placed in colcemide after removal of hypostome and tentacles or peduncleand basal disc show no signs of reconstituting these missing regions within48 h. Untreated control animals reconstitute both distal and proximal regionswithin 36 h at 26 °C. Colcemide-treated animals, after removal from colcemide,reconstitute missing regions within 48-120 h as described below.

Hydra treated with colcemide react in a characteristic manner. After 24 hall animals are extremely swollen and if punctured with a needle deflate likea pricked balloon, often spewing out masses of loose endoderm cells. Manyanimals are spherical at 24 h, but after 48 h most have assumed an irregularshape.

The polarity of hydra which have had hypostome and tentacles removed isusually unrecognizable after 24 h in colcemide, but the body wall of sphericalanimals often shows a characteristic pattern, being colourless and transparentat the poles as compared with the equator which is dense, pink and translucent.In some cases this pattern can be seen to represent the original linear axis, asindicated by debris characteristic of the basal disc still attached to one pole.

If the hypostome and tentacles are not removed, but the animals are woundedby a small lateral incision or by the removal of proximal regions, the originalpolarity is still recognizable after 24 h, but the tentacles are considerablyreduced in size; after 48 h the tentacles have disappeared completely andpolarity is in general no longer recognizable (Plate 1, fig. 1). The body of suchanimals often has a characteristic shape; longitudinal depressions appear so

184 G. WEBSTER

that when viewed from one pole the animals have a lobed appearance, thenumber of lobes being generally equal to the original number of tentacles(five or six). Buds at a medium stage of development often complete develop-ment while still in colcemide and may separate from the parent.

All colcemide-treated animals rapidly become enclosed in a gelatinousenvelope which becomes thicker during the period in colcemide and afterremoval. Microscopic examination reveals this envelope to consist mainly ofcytolysed cells, particularly cnidoblasts with nematocysts in all stages ofdifferentiation. It is interesting to note that when hydra subsequently reconstitutetentacles these are entirely or partially deficient in nematocysts in about 50 %of the animals.

Puromycin is toxic and concentrations of 1-1-1-7 x 10~4M (50-75 ju-g/ml)were the highest that could be employed. Even at these concentrations a pro-portion of the animals was killed after 24-48 h treatment. Reconstitutionwas not completely inhibited, and after 24 h some animals showed signs ofreconstituting missing peduncles and tentacles.

Puromycin does not produce the striking morphological changes seen incolcemide-treated animals. Intact hydra placed in 1-1 x 10~^ M puromycin showsigns of tentacle and peduncle regression after 24 h, as do those placed incolcemide, but even after 48 h these regions have not completely disappearedand most animals are still axiate with recognizable polarity. Buds often continueto develop and new ones may arise while the animals are still in puromycin.

After removal from puromycin, tentacle reconstitution occurs fairly quickly—within 24-48 h—but missing proximal regions are often reconstituted veryslowly, particularly in animals which have been treated for 48 h.

Table 1. Effect of colcemide on hydra possessing hypostome and tentacles

Piece treated

Whole animal (wounded)Animal minus peduncleand basal disc

No. ofanimals

2050

Time incolcemide

(h)

2448

Numberwith

multipledistal

structures

00

No. deador not

reconstituted

01

Effect of colcemide on hydra from which hypostome and tentacles are not removed

Animals which are wounded or have had proximal regions removed (i.e.basal disc and peduncle) and are treated with colcemide for up to 48 h andthen transferred to ' M ' reconstitute the missing regions and are exclusivelymonopolar in form. They possess one distinct group of tentacles, located atone end of a more or less linear axis (Table 1).

J. Embryo/, exp. Morph., Vol. J8, Part 2 PLATE 1

G. WEBSTER

Effect of colcemide on Hydra polarity 185

Effect of colcemide on hydra from which hypostome and tentacles are removedprior to treatment

When animals which have had the hypostome and tentacles removed (orthese regions together with peduncle and basal disc) are treated with colcemidefor 24-48 h and are then transferred to ' M', some show signs of tentaclereconstitution within 48 h. Most of these reconstituting animals possess onelocalized group of 4-6 tentacles in one region of a more or less linear axis; thesebecome typical monopolar forms. The remaining animals, which in generalreconstitute tentacles more slowly, include a significant number which shownot one localized group of tentacles but two or more distinct groups, or alterna-tively, in a few cases, an apparently random arrangement of isolated tentacleson an irregularly shaped body. Many of these animals regulate within 48 h.For example, two adjacent but apparently distinct groups of tentacles maybecome confluent and a typical monopolar animal is formed, often with branchedtentacles if the latter have fused at their bases. Alternatively two widely separatedgroups of tentacles will each acquire a distal axis and a Y-shaped, V-shaped orlinear bipolar form will result (Plate 1, figs. 2-4). Some animals, particularlythose showing an apparently random tentacle distribution, show little sign ofregulation and retain their irregular appearance for as long as 14 days.

It must be emphasized that in general only distal structures, i.e. tentaclesand distal axes, are involved initially in multiplication. In only eight animalswere multiple basal discs or peduncles observed and careful study indicatedthat such forms usually arose when the tentacle group formed in the middleof a long piece, thus leaving two free ends which became peduncles and basaldiscs (Plate 1, fig. 6).

The further fate of animals possessing multiple distal structures is variable.As noted above, extremely irregular animals usually undergo little change;

PLATE 1

Fig. 1. Intact hydra which have been treated with colcemide for 36 h; they have roundedup and the tentacles have been resorbed. The axial polarity is no longer visible.Fig. 2. Y-shaped bipolar, produced by treating a hydra minus hypostome and tentacles withcolcemide for 48 h. A basal disc is present (arrow). The thin and transparent appearance ofthe tentacles is due to the absence of nematocysts.Fig. 3. Linear bipolar produced by treating an isolated digestive zone with colcemide for24 h. No basal disc is present.Fig. 4. V-shaped bipolar produced by treating an isolated digestive zone with colcemidefor 24 h. A basal disc has developed at the apex of the V (arrow).Fig. 5. Hydra with multiple distal structures produced by treating an isolated digestive zonewith colcemide for 48 h. Three groups of tentacles can be seen (arrows).Fig. 6. Irregular bipolar produced by treating an isolated digestive zone with colcemidefor 48 h. Three basal discs (arrows) are present at the ends of the two shared axes; the twotentacle groups developed earlier at sites near the middle of an irregularly shaped longpiece.

186 G. WEBSTER

Y-shaped bipolars usually develop a basal disc at the foot of the Y within 48 hof reconstituting tentacles (Plate 1, fig. 2) and subsequently behave like bipolarsproduced by grafting a hypostome into the digestive zone of a host animal, i.e.the two axes show no sign of separating for up to 14 days. V-shaped or linearbipolars subsequently form a basal disc at the apex of the V or in the middleof the linear axis (Plate 1, fig. 4), and in many cases separation of the two axesoccurs within 7-14 days. The behaviour of these forms is analogous with thatof bipolars produced by grafting two hydra together with opposite polarity.

Table 2. Effect of colcemide on hydra from which the hypostome andtentacles are removed prior to treatment

Piece treated

Animals minus hypostomeand tentacles only

Untreated controls

Isolated digestive zones

Untreated controls

Isolated pedunclesUntreated controls

No. ofanimals

22

40

200

5020

200

20

20

Time incolcemide

(h)

24

48

—

2448

—

24

—

No. withmultiple

distalstructures

3(12%)

9(22%)0

14(28%)9(45%)0

0

0

No. deador not

reconstituted

1

0

0

00

0

2

0

The number of animals showing multiplication of distal structures is affectedby both the size of the treated piece and the duration of colcemide treatment(Table 2). Treatment of hydra minus hypostome and tentacles for 24 h usuallyresults in a small number (12 %) of the animals reconstituting multiple distalstructures which nearly all regulate to form Y-shaped bipolars with two distinctgroups of tentacles, two distal axes and a common proximal axis and basaldisc. Increasing the time of treatment of animals minus hypostome and tentaclesto 48 h results in an increase in the number of forms showing multiplicationof distal structures (22 %), but again the majority regulate to form Y-shapedbipolars. Reducing the size of the piece by employing isolated digestive zonesand treating for 24 h results in an increased number of animals with multipledistal structures (28 %), and these in general regulate to form V-shaped orlinear rather than Y-shaped bipolars. Treatment of isolated digestive zones for48 h with colcemide results in greatly retarded tentacle reconstitution; someanimals take up to 5-6 days before producing tentacles, but most animalseventually do so. More than 40 % of these animals show multiple tentaclesites and in a few cases they are apparently randomly distributed over anirregularly shaped body. Regulation to produce distinct distal axes does not

Effect of colcemide on Hydra polarity 187

usually take place in these very irregular forms. Animals which show lessdrastic alteration regulate to form V-shaped or linear bipolars.

In contrast to the behaviour of isolated digestive zones, peduncles whenisolated and treated with colcemide for 24 h reconstituted to produce exclusivelymonopolar forms.

Effect of colcemide on hydra from which the hypostome and tentacles are removedsubsequent to treatment

When intact animals are treated with colcemide for 24 h, removed fromcolcemide and the digestive zone isolated (i.e. hypostome and tentacles, buddingzone, peduncle and basal disc removed) they reconstitute to produce exclusivelymonopolar forms (Table 3 (1)). Animals which are treated with colcemide for24 h after removal of the peduncle and basal disc and then have the hypostomeand tentacles removed subsequent to colcemide treatment behave in a similarfashion; out of fifty animals treated, only one reconstituted to form a linearbipolar with a tentacle group at each end of the long axis (Table 3 (2)).

Table 3. Effect of colcemide on hydra from which the hypostomeand tentacles are removed subsequent to treatment

No. withTime in multiple No. dead

Piece isolated after No. of colcemide distal or nottreatment animals (h) structures reconstituted

Digestive zone (1) 32 24 0 5Digestive zone (2) 50 24 1(2%) 17

It should be emphasized that there are certain technical difficulties in thisexperiment. As described above, animals which have been treated with colcemidefor 24 h are considerably swollen and have partially or totally resorbed theirtentacles. For this reason it is extremely difficult to be certain that all thehypostome and tentacles have been removed, since these regions are distortedand spread over a considerable area of one pole of the spherical animal. In aneffort to ensure removal of all of these regions rather large amounts of tissuewere usually cut away and many of the pieces were therefore considerablysmaller in size than in the other experiments. This may account for the factthat a large number of animals failed to reconstitute tentacles in one of theseexperiments which employed a smaller piece (animal minus peduncle andbasal disc) to start with. The single animal showing multiple distal structuresmay therefore simply be a result of a failure to remove completely all of thehypostome and tentacles, in which case the animal would be expected toreconstitute in a similar manner to those from which the hypostome and tentaclesare not removed subsequent to colcemide treatment.

188 G. WEBSTER

Effect of puromydn on hydra from which the hypostome and tentacles areremoved prior to treatment

Isolated digestive zones when treated for 24-48 h with 1-1 x 10"4 M puromycinreconstitute to form typical monopolar hydra (Table 4). One animal from the24 h group produced a secondary distal axis bearing a single tentacle (verysimilar to a type 3 induction—Webster & Wolpert, 1966) giving rise to a Y-shapedbipolar.

Treatment of isolated proximal and distal halves of the digestive zone witha slightly higher concentration of puromycin (1-7 x 10~4M) resulted in a smallincrease in the frequency of bipolar reconstitution (Table 4).

Bipolars produced from the distal half of the digestive zone were all Y-shapedof exactly the same type as the one described above. The single one producedfrom the proximal half was a linear bipolar with two distinct tentacle groupsat opposite ends of the long axis.

Table 4. Effect of puromycin on hydra from which the hypostome andtentacles are removed prior to treatment

Piece treated

Isolated digestive zone

Distal half of digestive zoneProximal half of digestive zone

No. ofanimals

20202020

Time inpuromycin

(h)

24482424

No. withmultiple

distalstructures

1 (5%)04(20%)

1(5%)

No. deador not re-constituted

167

17

Thus, although puromycin has similar effects to colcemide in causing treatedanimals to reconstitute multiple distal structures, it is definitely not as effectiveas the latter substances. Both a higher concentration of chemical and a smallersize of treated piece are necessary before animals with multiple distal structuresare produced in significant numbers.

Effect of colcemide on hypostome formation and on the organizing properties ofthe hypostome

The effect of colcemide on the formation of a new hypostome was tested bytransplanting the distal tip of a reconstituting animal to the digestive zone ofan intact host hydra (Webster & Wolpert, 1966). Animals which had beenreconstituting in colcemide (5 x 10~5 M) for 8-9 h showed no signs of hypostomeformation from the subhypostomal region, though the majority of controlanimals possessed determined hypostomes at this time (Table 5).

The organizing ability of the hypostome was unaffected by colcemide treatment. Fragments of hypostome taken from hydra which had been treated (in

Effect of colcemide on Hydra polarity 189the absence of peduncle and basal disc) for 16-17 h induced secondary axesin the majority of the hosts to which they were transplanted (Table 5). Themorphological changes and the consequent obliteration of visible polaritywhich result from colcemide treatment prevent this experiment being performedon animals which have been treated for longer periods.

Table 5. Effect of colcemide on the formation and organizingproperties of the hypostome

Source of graft

Subhypostome

Hypostome

Time aftercutting when

grafted(h)

8-98-9

—

Time incolcemide

(h)

8-9—

16-17

No. ofsuccessful

grafts

1810

19

No. ofanimals

withsecondary

axis

08

17

Effect of colcemide and puromycin on the determined hypostome

Subhypostomal regions were isolated and allowed to reconstitute in ' M 'for 8 h. At this time, transplantation of pieces to the digestive zone of intacthost hydra indicated that a determined hypostome was present in the majorityof cases (Table 6). The remaining pieces were transferred to colcemide (5 x10~5 M) or puromycin (1-1 x 10~4 M) for 16 h, when transplantation experimentswere again performed. Treatment for this time in either substance does notresult in irreversible changes or death; washed pieces transferred to ' M ' allreconstituted tentacles within 24-48 h. It is important to note that about 50 %of the pieces which had been treated with puromycin possessed small tentaclebuds at the time of transplantation—clear evidence of the presence of a functionalhypostome.

The results shown in Table 6 indicate that colcemide treatment has no effecton the determined hypostome, which induced secondary axes in the majorityof cases. This confirms the results previously obtained with the 'adult' hypo-stome. Pieces treated with puromycin, however, induce secondary axes in avery few cases only, the majority of the grafts being absorbed. This indicatesthat puromycin treatment interferes with the ability of the determined hypo-stome to resist the influence of the factors which bring about absorptionfollowing transplantation (Webster, 19666). It is interesting that one puromycin-treated graft which did not induce was not absorbed, but transformed intoa small peduncle complete with basal disc, a striking indication of a changein properties to those of more proximal regions.

190 G. WEBSTER

Effect of colcemide and puromycin on the properties of the subhypostomal region

Freshly isolated subhypostomal regions were treated with colcemide (5 x10~5 M) or puromycin ( I - I X I O ^ M ) for 9h and then transplanted to thedigestive zones of host hydra from which the hypostome and tentacles hadbeen removed—the usual test for subhypostomal 'time-threshold' properties(Webster, 1966#, b). Treatment for this time does not produce irreversiblechanges or injury; washed pieces transferred to ' M ' all reconstituted tentacleswithin 24 h.

Table 6. Effect of colcemide and puromycin on the determined hypostome

Treatment

NoneColcemidePuromycin

Period whentreated

(h)

—

8-248-24

Time aftercutting when

grafted(h)

82424

No. ofsuccessful

grafts

152020

No. ofanimals with

secondaryaxes

10(66%)16(80%)3(15%)

Table 7. Effect of colcemide and puromycin on the propertiesof the subhypostomal region

Treatment

NoneColcemidePuromycin

Period whentreated

(h)

—0-90-9

Time aftercutting when

grafted(h)

099

No. ofsuccessful

grafts

101820

No. ofanimals with

secondaryaxes

6(60%)1 (6%)0

Puromycin treatment resulted in all the grafts being absorbed. In the case ofgrafts treated with colcemide, only one case of induction of a secondary axiswas observed. Control grafts (untreated) made immediately after isolationbehaved in the usual way and produced secondary axes in the majority ofcases (Table 7).

The results indicate that treatment of the subhypostomal region with colce-mide or puromycin changes the 'time-threshold' properties of this region tothose characteristic of more proximal regions (Webster, 1966a, b). The resultsalso, of course, confirm that colcemide inhibits hypostome formation. Whetherpuromycin acts in a similar fashion cannot be determined since, as the previousexperiment showed, puromycin intereferes with the resistance of the hypostometo factors bringing about absorption.

Effect of colcemide on Hydra polarity 191

DISCUSSION

The results show that colcemide treatment of hydra from which the hypostomehas been removed can bring about an alteration of polarity such that multipledistal structures, i.e. more than one hypostome, can form in a single animal.The alterations observed were variable and resulted in the production of a rangeof forms, from animals showing bipolar organization to those in which hypo-stomes were more or less randomly distributed. Pretreatment with colcemidefollowed by removal of the hypostome resulted in only one animal reconstitutingmore than one hypostome. In no case did such forms arise if the originalhypostome was not removed. Treatment with puromycin in the absence of thehypostome was much less effective in altering polarity as judged by subsequentreconstitution than was treatment with colcemide. The effects of colcemide andpuromycin on 'physiological' polarity were paralleled by their morphologicaleffects; visible polarity was completely obliterated in colcemide treated animalsbut persisted in those treated with puromycin.

The transplantation experiments indicated that hypostome formation wasinhibited by colcemide but that this substance did not irreversibly affect thedetermined hypostome or the 'adult' hypostome, which retained their organ-izing properties. The results of puromycin treatment are curious. Hypostomeformation was not inhibited completely, as indicated by the fact that someanimals reconstituted tentacles while in puromycin. However, the hypostomewhich was formed did not possess the normal resistance to absorption, asshown by the transplantation experiments. Both colcemide and puromycinchanged the ' time-threshold' properties of the subhypostomal region to thosecharacteristic of more proximal regions.

The simplest way of explaining the effects of colcemide and puromycin onregulation is to postulate that treatment with these substances flattens orabolishes the disto-proximal axial gradient in 'time-threshold' properties(Webster, 1966 a), so that when released from inhibition distal regions have noadvantage over proximal regions as regards hypostome formation. The resultsof the experiments on the subhypostomal region are direct evidence that treat-ment with either substance changes the 'time-threshold' properties of this regionto those characteristic of more proximal regions, i.e. regions which are lower onthe gradient. In order that the gradient be flattened, it is clear that distal regionsmust be affected more than proximal regions. No evidence is available on thispoint, though the fact that colcemide treatment of isolated peduncles did notresult in the formation of multiple distal structures may indicate that proximalregions are less affected. This is also suggested by the fact that animals fromwhich the hypostome and tentacles only have been removed (i.e. animals possess-ing a peduncle) produced mainly Y-shaped bipolars with a hypostome at eachend of the digestive zone. Kanatani (1958) observed that in planaria bipolarreconstitution was more frequent from anterior than from posterior regions.

192 G. WEBSTER

Consider what would happen when an animal whose hypostome has beenremoved is placed in colcemide. New hypostome formation is prevented andthe level of inhibition will fall, presumably throughout the animal. The gradientin ' time-threshold' properties is flattened. On removal from colcemide, all regionswill begin forming hypostome, and those with the 'highest' 'time-threshold'properties will succeed and inhibit those with 'lower' properties. The finalresult will be variable and animals will form one or more hypostomes, dependingon how the gradient has been altered and how the developing hypostomesinfluence each other. A hypostome, once formed, will immediately begin toraise the level of inhibition in its immediate neighbourhood (Webster, 19666).It is probable that any other hypostome which forms will be as far away fromthe first as possible, since we know that the effectiveness of inhibition decreaseswith distance (Webster, 1966a). For example, in an isolated digestive zone thenew hypostomes will form at opposite ends and a linear bipolar will result.

When the original hypostome was left in place during colcemide treatmentand subsequently removed, multiplication of distal structures was observed inonly one animal out of those which reconstituted. It will be remembered thatthere were technical difficulties in this experiment, and the possibility that thehypostome was not completely removed in all animals makes it unwise toconclude that this procedure is less effective in altering polarity. However, it ispossible that leaving the hypostome in situ during treatment tends to counteractthe action of colcemide, and this is consistent with the idea that the hypostomeplays some role in controlling the gradient in 'time-threshold' properties(Webster, 19666).

In those experiments in which the hypostome was left in place both duringand subsequent to colcemide treatment no multipolar forms were produced.Even if the gradient is flattened in these cases, we have seen that the determinedhypostome is not irreversibly affected by colcemide treatment, so that, onremoval from the substance, a functional hypostome is present which canpresumably inhibit hypostome formation elsewhere in the animal. Even if theinhibitory action of the hypostome is impaired by colcemide treatment, sup-pression of new hypostome formation will occur at a low level of inhibitionbecause of the depression of thresholds.

Puromycin is much less effective than colcemide in causing the productionof multipolar forms, even though it has a similar effect to the latter substanceon 'time-threshold' properties. This is probably due to the fact that puromycindoes not inhibit the formation of a hypostome in all animals. An animal(minus hypostome) when placed in puromycin may develop a new hypostomewhich, although abnormal in resistance to absorption when transplanted, isadequate to inhibit further hypostome formation in situ. Thus reconstitutionwill be monopolar.

It is interesting to note that colcemide treatment produced about twice asmany multipolar forms from isolated digestive zones as compared with larger

Effect of colcemide on Hydra polarity 193pieces (animals minus hypostome and tentacles only). Similar results wereobtained with puromycin. This seems to be a general feature of bipolar re-constitution. For example, in marine hydroids the frequency of spontaneousbipolar forms is higher in small pieces than in large pieces (Child, 1941).A completely satisfactory explanation of this result is not at present possible. Itis clear that the two ends of a small piece will have fairly similar' time-threshold'properties so that it is easier for them to become identical than for the twoends of a large piece. However, this explanation will not account for the differencein numbers between small and large pieces, unless the additional assumptionis made that a hypostome has a greater tendency to arise from a cut surface(i.e. an end) than from an intact area.

Although colcemide and puromycin have similar effects on the' time-threshold'properties of the subhypostomal region, their effects on hypostome formationand the determined hypostome are strikingly different. Colcemide appears toblock hypostome formation completely, but the determined hypostome isresistant to and is not irreversibly affected by colcemide treatment. The behaviourof the determined hypostome with respect to colcemide is reminiscent of itsbehaviour with respect to the 'natural' inhibitor of hypostome formation(Webster, 1966a).

The effect of puromycin is extremely interesting. This substance does notappear to inhibit hypostome formation since organizing properties may beacquired in some cases (as judged by the production of tentacles). However, itseems to interfere with the resistance to absorption of the determined hypostome,and presumably with the acquisition of resistance during hypostome formation.This suggests very strongly that organizing ability and resistance to absorptionare quite distinct properties. If the resistance to absorption normally displayedby the determined hypostome is dependent on resistance to inhibition andtherefore upon high threshold, the fact that organizing properties can beacquired and retained in the absence of resistance suggests that the observedrise in threshold during hypostome formation (Webster, 1966 b) might not bea necessary part of the process of hypostome formation. This has importantconsequences for our understanding of how release from inhibition occursduring normal regulation. If hypostome formation can occur without rise inthreshold, then release from inhibition must result from a fall in the level ofinhibition.

If the resistance to absorption displayed by the hypostome is dependent onthreshold, then its threshold properties must be less labile than those of non-hypostomal regions since we have seen that subhypostomal threshold propertiesare altered by both colcemide and puromycin but hypostomal resistance isunaffected by colcemide. It is, of course, possible that higher concentrationsof colcemide might interfere with hypostomal resistance. It is known that thethreshold properties of non-hypostomal regions are unstable and change asa consequence of isolation or transplantation. The hypostome, however, retains

194 G. WEBSTER

its characteristic properties following transplantation and for as long as 7 daysafter isolation (Webster, 19666, and unpublished observations).

An alternative explanation is possible for the different results obtained withcolcemide and puromycin if it is assumed that colcemide has no direct effectupon 'time-threshold' properties and that these change as a result of the inhibitionof hypostome formation. It is known that the 'time-threshold' properties ofa region are determined by the position of the region on the linear axis inrelation to the hypostome. Changing the position of a region so that it isnearer the hypostome 'raises' the 'time-threshold' properties; moving it fartheraway from the hypostome' lowers' the' time-threshold' properties. These changesonly occur if the level of inhibition remains above the threshold so that hypo-stome formation is prevented (Webster, 19666). It seems reasonable to postulatethat if a region is removed to an 'indefinite' distance from a hypostome, i.e.isolated, and at the same time prevented from forming hypostome by colcemide,then 'time-threshold' properties would 'fall'. This explanation is consistent withthe observations that colcemide has little effect on the polarity of animalstreated prior to hypostome removal and that the determined hypostome isresistant to treatment. Puromycin, on the other hand, would be supposed tohave a direct effect on 'time-threshold' properties and hence could affect bothhypostomal and non-hypostomal regions. Its relative inefficiency at bringingabout alteration of polarity would result from its failure to inhibit hypostomedetermination. This explanation could be subjected to experimental test byexamining the 'time-threshold' properties of regions of animals treated withcolcemide in the presence of the hypostome. It might be mentioned that inplanaria polarity can be altered in animals treated prior to head removal(Kanatani, 1958; Flickinger & Coward, 1962).

The present results are of interest in relation to those of Lesh & Burnett(1964, 1966) and Lentz (1965). These workers demonstrated that a substancewas present in homogenates of hydra which when applied to isolated piecesof the digestive zone for 4 h was capable of altering polarity and producinganimals with multiple distal structures. Lesh & Burnett have claimed that thissubstance is identical with the so-called 'growth-stimulating principle', postu-lated by Burnett (1961) to account for distal regeneration in hydra, and alsothat the substance is 'responsible for polarity'. The forms produced as a resultof treatment with the homogenate appear to be identical with those producedin the present experiments as a result of colcemide treatment. Colcemide is aninhibitor of protein synthesis in planaria (Flickinger, 1959) and of mitosis ina variety of organisms (Schar, Loustalot & Gross, 1954) including hydra(Webster, 1964); it can therefore fairly be described as a 'growth-inhibitingsubstance'. The fact that it appears to have identical effects to the substancepresent in hydra homogenates suggests that the conclusion that the latter isa 'growth-stimulating substance' is unjustified and also suggests that 'growth'plays no role in the control of polarity. More important, colcemide inhibits

Effect of colcemide on Hydra polarity 195hypostome formation and the alterations of polarity which are produced(involving effects on regional 'time-threshold' properties) take place in theabsence of a hypostome. In other words, hypostome formation as such is nota necessary feature of any alteration in polarity but merely reveals at a laterstage that such alterations have taken place. In the experiments of Lesh andBurnett alteration of polarity is said to occur as a result of the actual stimulationof hypostome and tentacle formation. It is worth noting that these workerstreated isolated pieces for only 4 h. If the substance they are dealing with isreally 'growth-stimulating' then continuous treatment during regenerationshould have the same or a greater effect than brief exposure. Also, the substanceshould be effective irrespective of the presence or absence of the hypostome.These critical experiments do not appear to have been done. In the light of thesecriticisms it must be concluded that the significance for normal regulation ofthe substance present in hydra homogenates is obscure.

SUMMARY

1. The effect of colcemide and puromycin on polarity and regulation inhydra has been investigated at the biological level using isolation and trans-plantation techniques.

2. Colcemide treatment of hydra from which the hypostome has been removedcan bring about an alteration of polarity so that multiple distal structures(hypostome and tentacles) can form on a single animal. Such forms do notarise if the original hypostome is not removed. Treatment with puromycin ismuch less effective in altering polarity.

3. Transplantation experiments indicate that hypostome formation is in-hibited by colcemide treatment but that this substance does not irreversiblyaffect the determined hypostome or the 'adult' hypostome which retain theirorganizing properties.

4. Puromycin treatment does not inhibit hypostome formation completelysince some animals reconstitute tentacles while in puromycin. However, thehypostome which is formed does not possess the normal resistance to absorptionfollowing transplantation. This result may indicate that the factors responsiblefor the organizing properties of the hypostome (i.e. tentacle induction) aredistinct from those which control resistance to absorption.

5. Both colcemide and puromycin change certain important properties ofthe subhypostomal region to those characteristic of more proximal regions.These properties are believed to 'control' polarity.

6. The effects of colcemide and puromycin on polarity and regulation arediscussed in terms of their effects on a control system which has been previouslypostulated to account for polarised regulation in hydra. This system involvesaxial gradients in time for hypostome determination, inhibition of hypostomeformation and threshold for inhibition. The experimental results can be explainedas a consequence of the direct or indirect action of these substances on theaxial gradients.

196 G. WEBSTER

RESUME

Etudes sur la regulation chez VHydre. IV. Action de la colcemideet de la puromycine sur la polarite et la regulation

1. L'action de la colcemide et de la puromycine sur la polarite et la regulationchez l'hydre a ete etudiee au niveau biologique au moyen de techniquesd'isolement et de transplantation.

2. Le traitement a la colcemide d'une hydre dont on a coupe l'hypostomepeut determiner un changement de polarite tel que de multiples structuresdistales (hypostome et tentacules) peuvent se former sur un seul animal. De tellesformations n'apparaissent pas si l'hypostome primitif n'est pas supprime. Letraitement a la puromycine affecte beaucoup moins la polarite.

3. Des experiences de transplantation montrent que la formation de l'hypo-stome est inhibee par un traitement a la colcemide mais que cette substancen'affecte pas irreversiblement l'hypostome determine ou l'hypostome 'adulte'qui conservent leurs proprietes organisatrices.

4. Le traitement a la puromycine n'inhibe pas completement la formationde l'hypostome puisque quelques animaux reconstituent des tentacules pendantce traitement. Cependant, l'hypostome qui est forme ne possede pas la resistancenormale a l'absorption qui suit la transplantation. Ce resultat peut indiquerque les facteurs responsables des proprietes organisatrices de l'hypostome(c'est-a-dire 1'induction des tentacules) sont distincts de ceux qui controlent laresistance a l'absorption.

5. La colcemide et la puromycine transforment certaines proprietes impor-tantes de la region sous-hypostomale en proprietes caracteristiques des regionsplus proximales. On pense que ces proprietes 'controlent' la polarite.

6. Les effets de la colcemide et de la puromycine sur la polarite et la regulationsont discutes en fonction de leur action sur un systeme de controle qui a eteprecedemment postule pour expliquer la regulation polarisee chez l'hydre.Ce systeme implique des gradients axiaux dans le temps pour la determinationde l'hypostome, l'inhibition de la formation de l'hypostome et le seuil del'inhibition. Les resultats experimentaux peuvent etre interpreted comme uneconsequence de l'action directe ou indirecte de ces substances sur les gradientsaxiaux.

I should like to thank Dr L. Wolpert for helpful discussions during the course of thiswork, and the Agricultural Research Council for a Postgraduate Research Studentship.

Effect of colcemide on Hydra polarity 197

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(Manuscript received 6 January 1967)

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