Positional Information and Positional Signalling in Hydra

AMER. ZOOL., 14:647-663 (1974).

Positional Information and Positional Signalling in Hydra

L. WOLPERT, A. HORNBRUCH AND M. R. B. CLARKE

Department of Biology as Applied to Medicine, The Middlesex Hospital MedicalSchool, London W1P 6DB, United Kingdom and University of London,

Institute of Computer Science, London WCl, United Kingdom

SYNOPSIS. A model is pioposcd for regulation and regeneration of the head end of hydrain terms of positional information, which involves two gradients. One is a diffusiblesubstance made at the head end, which may be regarded as a positional signal. Theother is a more stable cellular parameter which is the positional value. The rule forhead end formation is that the concentration of the diffusible substance falls athreshold amount below the positional value. This model, for which some computersimulation is provided, can account for head end formation in a wide variety of grafts.Evidence for a diffusable signal is provided by experiments in which the time/distancerelationships for head inhibition by a grafted head are determined. Changes in posi-tional value during regulation have been assayed and are much slower away from theboundary. Polarity is interpreted in terms of the interaction between the two gradients.The biochemical basis of the gradients is not known, but an approach to the problemhas been made by treating hydra with a variety of chemical agents.

Hydra shows remarkable capacity forregeneration and regulation when piecesare removed or added (Webster, 1971).The idea that gradients may be involvedin such processes, which occur in numer-ous developmental systems, is a very oldone and emphasized by Child (1941) inparticular. There has nevertheless been, inthe past, a singular absence in develop-mental biology of attempts to constructquantitative or rigorous models or theoriesof how gradients could be related to regu-lation, and there is a legacy of ratherunsatisfactory concepts such as dominance,regulation and gradient fields. One of theaims in proposing the concept of posi-tional information was to introduce amore rigorous and quantitative conceptualframework for considering pattern forma-tion and regulation (Wolpert, 1969, 1971).The basis of the mechanism is that cellsfirst have their position specified with re-spect to certain boundary regions, as in acoordinate system, and then interpret thispositional information according to theirgenome and developmental history. Theprocess of interpretation will often involve

This work is supported by the NuffieldFoundation.

cytodifferentiation. Once the idea of posi-tion specification is accepted, and this isan idea explicitly put forward by Driesch(1908), then attention is immediatelydrawn to the specification of the boundaryregions, and the scalar and vectorial prop-erties of the system which enable positionto be specified. Regeneration in these termsis the re-establishment of the co-ordinatesystem when a part is removed, that is,the re-establishment of boundary valuesand new positional values in relation tothem.

In terms of positional information ahydra may be viewed, along its main axis,as a bipolar field (Wolpert et al., 1971).A field is defined as that set of cells whichhave their position specified with respectto the same coordinate system or boundaryregions. The field is bipolar since the twoboundary reference regions appear to bethe head and foot ends. The variation inpositional value along the hydra may berepresented by a gradient in some cellularparameter P which decreases monotoni-cally from the head end to the foot end.In order that this gradient provide posi-tional information, the value to the headand foot ends must be fixed and the valuesre-established when a head or foot is re-

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648 WOLPERT, HORNBRUCH, AND CLARKE

100.

80.

60.

40.

20.

0

cut cut cut

100.

80.

60.

4a

20.

0

4

FIG. 1. Positional information may be representedin hydra by a gradient in some parameter P as ina. The value at the head end is a and at the footend a- If the system is cut as indicated, the posi-tional gradient would regulate so as to re-establishthe boundary conditions, but the slope of the

moved (Fig. 1). Another way of providingpositional information would be to havetwo gradients, P and P', the ratio of whichcould be used. In either case regenerationwould involve the re-establishment of theboundary regions and the gradients whenthe part is removed. In hydra, this newboundary, in simple regeneration, wouldbe at the cut surface since regeneration isby morphallaxis. There is now substantialevidence that cell growth and division playno major role in regeneration and budding(Clarkson and Wolpert, 1967; Wolpertet al., 1971; Hicklin and Wolpert, 19736).Much of this paper will be concerned withpossible mechanisms whereby the boundaryvalues are established and localized.

It is a basic assumption that the ob-

gradient would increase. Positional informationcould be provided by two gradients as in b. Inthis case regulation following cutting would againlead to re-establishment of the boundary valuesbut the slope of P and P' would be unaltered.

served form of hydra results from the cellsinterpreting their positional value. Cellsform tentacles or a hypostome becausethey are at a particular level in the gradi-ent. The form thus results from the cells'interpretation of positional information.Unfortunately this is a very weak link sincethe cellular activities involved in, for ex-ample, tentacle formation, are not known,but hopefully may involve changes in cellcontact and motility (Campbell, 1968) ina manner similar to that postulated forsea urchin morphogenesis (Gustafson andWolpert, 1963, 1967). Our ignorance ofthe cellular factors determining form ofhydra is severe, and it even remains amystery as to why hydra is a tube com-prising two cell layers.

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POSITIONAL INFORMATION IN HYDRA 649

In the considerations that follow, hydrawill be largely treated as a homogeneoustissue and no attention will be given tothe distribution of cell types as described,for example, by Bode et al. (1973).

In investigating positional informationin hydra, grafting provides a powerful,but limited, technique. The technique ispowerful since it is easy to do a largevariety of grafts. It is limited because oneassays the results almost entirely in termsof the formation of head and foot endswhich are the only reliable markers onehas. This can be contrasted with other sys-tems, such as the insect cuticle, where onemay have local markers throughout, e.g.,Lawrence (1970). However, as pointed outabove, in terms of positional information,the re-establishment of the boundary re-gions is a key event in regeneration.

FIG. 2. Photograph of hydra to show the regionsH1234B56F.

• For a large and varied number of graftswe have constructed a synthetic model. Wehave a convenient notation for the regionsof the axis in hydra (Fig. 2). The headregion, consisting of hypostome and ten-tacles, is represented by H, and the diges-tive zone by four equal-sized regions,/, 2, 3, 4; B designates the budding re-gion, 5 and 6 the distal and proximalhalves of the peduncle, and F the foot orbasal disk. In terms of this notation agraft of head and distal gastric regiononto the subhypostome of another animalis given as H12J12 . . . F. Before present-ing the model and the experiments it maybe convenient to summarize briefly someof the results (Webster and Wolpert, 1966;Webster, 1966aA 1971; Wolpert et al.,1971) with as little reference to a particu-lar model as possible, (i) The time re-quired to become a head increases with dis-tance from the head end; (ii) the headcan prevent other regions from forming ahead end, and this inhibition falls off withdistance; (iii) the "level" of inhibitor re-quired to inhibit a piece from forming ahead end—the threshold for inhibition—decreases with distance from the head end;(iv) the changes occurring during regen-eration are localized very close to the cutsurfaces; (v) new ends form more easilyat cut surfaces.

We have investigated the spatial andtemporal characteristics of these processesin some detail and the current model pos-tulates, with respect to head end regenera-tion, two gradients (Fig. 3). The firstgradient is considered to be of a diffusiblesubstance S, which, in the steady state, isproduced by a source at the head endwhich maintains S at a constant concen-tration there. S is of course broken downand we have assumed that this occurs onlyat the foot end, which acts as a sink forS and maintains it at a constant concen-tration there. This results in a linear gradi-ent of S along the animal. The secondgradient is of P, the positional value, whichis considered to be some cellular param-eter, and which is not diffusible. Changesin P are considered to occur only by syn-thesis and breakdown. The form of the

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H I 2 3 4 B 5 6 F

FIG. 3. Diagram to illustrate the main changes inS and P during normal regeneration. The intactanimal is shown in a. When the head (H) is re-moved the concentration of S falls as in b. Whenit reaches a critical value below P, P is synthesizeduntil the boundary value of 100 is reached (c) . Itthen becomes a source of 5 again (d) . The increaseof P away from the boundary is slow and is notshown.

gradient in P is taken, for simplicity, tobe identical in the equilibrium state tothat of S. The rule for initiating head endformation is that S must fall a thresholdamount below P. P then increases to itsboundary value and acts as a source of Sagain.

Before considering the experiments inmore detail, it may be helpful to indicatehow the model was arrived at. One seriesof experiments which showed some sort ofthreshold phenomenon to be involved wasthe absorption of region 1 if grafted inbetween regions 2 and 3 of an intact ani-mal, whereas if placed in the 56 region,it formed a head at the site of graft (Web-ster, 1966a). In terms of the model, thehigh level of S in the grafted 1 is beingreduced when placed in a level of lowconcentration of 5. If this is sufficientlylow, then head formation is induced. Thisimplies that there is a more stable gradient,not changed by grafting, with which S canbe compared. One can think of P as aparameter which tells the cell what it cur-

rently is, and 5 is a parameter which canrespond to the state of neighboring cells.It can be seen that the model has impor-tant similarities to that of Lawrence et al.(1972) for the insect epidermis.

Crick's (1970) emphasis on diffusion asa plausible mechanism of communicationencouraged us to make use of this to ac-count for one of the gradients, and thesimplest model we could think of involveda diffusible substance made at the head.When the head was removed, its concen-tration would fall, and at some criticalvalue, new head formation would be ini-tiated. From the theoretical point of viewit is necessary, in the first instance, to makesuch a model regenerate a head end boun-dary and only one head end boundary. Itis a non-trivial problem to construct amodel such that a new head end is re-stricted to a given number of cells at thecut surface and to match the graftingexperiments.

Our current model envisages the follow-ing sequence. Following the removal of thehead end the concentration of S falls, bydiffusion, until it is a critical concentra-tion below the original value as "remem-

I • •

M|1 \2 [3 J4 ,' B ; 5 ; 6 ;F|

MI i i i5i i i i i i r

FIG. 4a. Diagram of a 17-celled hydra which wasused for computer simulation of the changes in5 and P. The length of the hydra is taken as 3mm and the diffusion constant as 2 X HH em'/sec.

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IO0L

80.

60.

C * H . I 3 I 4 I 5 I 6 I 7 I 6 I I 3 | 4 | 5 | 6 | 7 | 8 I 9 T

• A A A A A C A A A A A A D A A A A A A A

Haw 1-5 375FIG. 4b. Simulation of regeneration of the beadend from region 1 following removal of H. The

bered" by P. We have used, arbitrarily, agradient going from 100 to 10. The hydrais 3 mm long and is divided for computersimulation into 17 cells (Fig. 4a). Wehave used a diffusion constant of 2 X 10"7

as this is a plausible estimate for a smallmolecule (Crick, 1970) and was the valueobtained from our own experimentalstudies on the time for a "signal" to betransmitted from the head end (Wolpertet al., 1972). The formation of a new headend boundary is triggered when 5 falls acritical value, say 10%, below P. At thisvalue synthesis of P starts and continuesuntil P is 100 again, but synthesis of Pcan be inhibited before P reaches 100 if Sincreases to within the critical value, again,perhaps by diffusion from some otherregion. When P reaches 100, it again actsas a source for 5.

We have carried out a simple computersimulation of this process of which Figure4b shows one example. Experimentally anew head end is determined from a 1region in 4 to 5 hr with H. littoralis at26 C. We have assumed that about one-third of this time is for S to fall and theremainder for P to rise; this will be dis-cussed in detail elsewhere (Clarke and

7 0 h*ara

cell states and the transition rules are shown inTable 1.

Wolpert, 1973). When S falls the criticalamount, 10%, below P, P is synthesized.In order to ensure that only the end cellbecomes determined as a head, it is neces-sary for this cell also to make S at a slowrate and so prevent further cells being"switched on" and becoming heads. S mustnot be made so fast, however, that it in-hibits synthesis of P, the synthesis of Pbeing inhibited if S rises to within 10%before P reaches 100. Only when P reaches100 does rapid synthesis of S occur to re-

TABLE 1. The behavior of P and S for different cellslates and the transition rules.

State

State

State

State

A

B

C

D

State

State

StateState

P

Fixed

dT = '

100

100

Rules for

A-> State B

B-> State A

B -> State CC—>State D

transitions

if 1 -

if 1 -

if P >if S >

dSdtd5dt

S .P :

S .P100100

s

Diffusing

= 5, (Slow)

= ks, (Fast)

100

> 0.1

<0.1

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establish the gradient. It will be noted thatthe changes in P are localized at the cutend in this model. A more sophisticatedmodel (Clarke and Wolpert, 1973) allowssome leakage of S into the external me-dium at the cut surface and will considerthe threshold and other factors in moredetail.

The regeneration of a hydra in terms ofthis model can be represented in terms ofcell states and their behavior with respectto S and P (Table 1). Away from theboundary, changes in P may involve a fol-low-up servomechanism (Wolpert, 1971)to re-establish the steady state linear gradi-ent, but this is not important in boundaryformation.

INSTANTANEOUS AXIAL CRAFTS

Instantaneous grafts, which are madeimmediately after cutting, are distinguishedfrom those in which the grafts are madeat various times after removing the sourceor the sink, i.e., H or F. The interest ininstantaneous axial grafts lies in whetheror not heads form at the junctions betweengrafts experimentally and according to themodel, that is, whether a head end willform when S falls significantly below P bydiffusion between cut ends. Some typicalexperimental results for H. littoralis areshown in Table 2 (Hicklin et al., 1973)and illustrated in Figure 5.

For comparison, some simple computersimulations of diffusion of S within thegrafts have been carried out using thehypothetical hydra of Figure 4. The effecton the distribution of S of a 12/12 . . . F

FIG. 5. Photograph of a graft resulting in a headforming at the junction.

graft is shown in Figure 6. The changesof S with time for some of the grafts inTable 2 are given in Figure 7.

Grafts of H12/12 . . . F do not experi-mentally give structures at the junction.The analogue in the model is that whileS falls below P at the junction it does notfall below the critical value. However, inboth H123/12 . . . F and 12/12 . . . F, ascan be seen from Figure la, S falls fur-ther below P and this can result in struc-tures forming at the junction. Of particu-lar interest is 12/56F where a head formsat the junction from the proximal end ofthe 2 region. No structures form in H12/56F and the fall in S is less (Fig. 7b). Ina later paper a more detailed analysis ofcomputer simulation will be given whichwill include consideration of the value of

TABLE 2. Formation of structures at the junction following axial grafting.

Graft

H12H12312H1212123412

Host

12 . . .12 . . .12 . . .56F56F34 . . .34 . . .23 . . .

FFF

FFF

Number

2021242048202120

Normal

1005213

10058

10029OS

Results

Heads

n42

7115

/o

Heads andfeet

•5'

m

o

Feet

4$11

25

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90.

80.

70.

60.

50.

2 I 1FIG. 6. The distribution oC S 1 hr ( • - • ) aftergrafting 12 onto 12 . . . F.

the threshold and the effect of a cut sur-face (Clarke and Wolpert, 1973).

A large number of other grafts such as12/34 . . . F and 23/23 . . . F have beencarried out and on the whole are consis-tent with this analysis (Hicklin et al.,1973).

Similar experiments on H. nttenuata giveresults along similar lines but which differon the whole quantitatively rather thanqualitatively.

FORMATION OF THE FOOT END

It would be very attractive if the inter-actions between 5 and P could also ac-count for foot end formation: for example,might it not be possible that foot endformation was initiated when the concen-tration of S rose a threshold amount aboveP? Unfortunately this simple symmetricalmodel cannot easily be sustained sincethere are a variety of grafts in which con-tradictory results are observed. For ex-ample, in grafts of H12/12 . . . F, S willrise at the proximal end of the distal 2region but not foot forms, whereas in12/12 . . . F, when S is lower there, a footoften forms. From a variety of such experi-ments we have concluded that a signalfrom the head end can inhibit foot endformation.

There is good evidence that the presenceof a foot can prevent the formation ofanother foot in adjacent regions (MacWil-

liams and Kafatos, 1968; MacWilliams etal., 1970). This ability of the foot end toinhibit foot formation is, of course, analo-gous to the properties of the head end, andthis analogy is further strengthened by ourresults (Hicklin et al., 1973) which showedthe symmetrical behavior of H12/12 . . . Fand H12 . . . 56/56F type grafts. It wouldseem that the simplest model to accommo-date the results would be to postulateanother gradient in a diffusible substanceS' which would be produced at the footend. Foot formation would then dependon S' falling to within a threshold amountof A-P where A is a constant greater thanthe maximum value of P. In addition, Swould tend to inhibit foot formation. Itwill be important to know if S' can alsoinfluence P. Is, for example, the forma-tion of regions 56, the peduncle, controlled

90.

to.

FIG. la. Change in 5 with time at distal end o£region 1 at the junction in grafts of the typeH12/12 . . . F. b, Changes in S with time at theproximal end of region 2 at the junction in graftsof. the type H12/56F.

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by a positional signal from the foot end?The interesting point to emerge from theseexperiments is that determination and de-velopment of the foot end appears to besymmetrical with that at the head end(Hicklin and Wolpert, 1973a). Like graftsfrom the head end, lateral grafts from thefoot end are capable of "inducing" ororganizing a secondary axis. This propertyis also shown by peduncle tissue graftedto the gastric region though not to thepeduncle.

The times for foot regeneration anddetermination are also similar to those forthe head end (see Webster and Wolpert,1966) . First, there is a gradient in the timerequired for the development of the footafter cutting depending on the distance ofthe regenerating end from the foot. A footwill form only very slowly from the cutproximal end of HI. This is comparableto the slow formation of the head in 6Fanimals (Webster and Wolpert, 1966).There is also a gradient in the time forfoot end determination as assayed by lateralgrafting. The times are short and deter-mination rapid. For peduncle tissue it isnot even possible to measure a time fordetermination since it will induce immedi-ately following lateral grafting, suggestingthat determination takes place within anhour. For region 4 the determination timeis only 3 to 4 hr. For head determination,the time required by the 3 region is about10 hr (Hicklin et al., 1973) and a similartime is required for foot determination.In view of the ability of region 5 to "in-duce" a proximal axis when grafted intothe gastric region, it was interesting toobserve the behavior of a 5 region whenit was regenerating the head: there was aswitch from proximal to distal inductionsat between 12 and 18 hr. It is perhapssurprising that the ability to induce proxi-mal axes had not been lost even after 12hr of regeneration.

We have obtained some further evidencethat the presence of the head tends toinhibit foot formation which supports ourprevious conclusion (Hicklin et al., 1973).A higher percentage of proximal induc-

tions was obtained with grafts from region5 when the host's head was removed thanwhen it was present.

These similarities of behavior exhibitedby the foot and head confirm our viewthat they are the boundary regions of abipolar positional field. These boundaryregions appear capable of organizing a newaxis, presumably by establishing a newboundary to the positional field. The headend may be dominant in so far as it mayinhibit foot end formation. One may ac-count for foot end formation in a similarway to head end formation: their sym-metrical behavior with respect to axialgrafting has already been established(Hicklin et al., 1973). What is far fromclear is whether they contribute to theassignment of positional information tothe intermediate regions and if so, whattheir relative contribution may be.

DYNAMICS OF HEAD END FORMATION

Using lateral grafts, Webster and Wol-pert (1966) found that a new H is de-termined at the level 1 about 5 hr afterremoval of the head. The axial grafts de-scribed in the previous section can also beused to obtain data on the dynamics ofhead end formation (Hicklin and Wolpert,1973c). The two operational questions onemay ask are how long it takes for a regionto change so that head inhibition is nolonger possible, and how long it takes fora region to be able to inhibit other regions.In terms of the model this means findingout how P and 5 change with time.

Since the combination H12J12 . . . Fdoes not give structures at the junction,one may ask how long a regenerating12 . . . F could be left before a H12 graftwould no longer inhibit. The results ofsuch an experiment are shown in Table 3.The time for 50% inhibition is 4 to 5 hr,which is similar to the time for lateralgrafting. For regenerating 34 . . . F thetime is about 10 hr which is consistentwith our earlier findings (Webster andWolpert, 1966) that the time required forhead end determination increases with dis-tance from the head end.

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TABLE 3. H 1 2 onto regenerating 12 . . . F : formation of structures at the junction.

Regenerating time(hours)

123456

Number ofcombinations

202414102312

Normal

20 (100%)23(96%)13(937o)7(70%)

0

Results

Heads

01 (4%)I (7%)3(30%)

16(70%)12(100%)

Heads and Feet

00007 (30%)0

An experiment to determine the time fora 12 to become a H12 with respect to in-hibition is shown in Table 4, the timebeing about 12 hr.

An important feature of the dynamicsof head end formation is the effect of thecut surface. Other considerations suggestthat it is more rapid there. Direct evidencewas obtained by examining head deter-mination by lateral grafting of region 1f r o m F . . . 21/12 . . . F and 12 . . . F. F o rthe former the time was about 8 hr, whichis significantly longer than the time of 4to 5 hr when the regenerating 1 is at thecut surface. The simplest interpretation ofthese results is that S can diffuse out ofthe cells into the external medium at thecut thus speeding up the fall in S.

SIGNALLING ALONG HYDRA

The experiments given above providegood evidence for some sort of a signalfrom the head end affecting head forma-tion in other regions. It seems that thesource of the signal at the head end isdistributed since removal of parts of thehead in H12/12 . . . F combinations leadsto structures forming at the junction(Hicklin et al., 1973). It also seems that

cell contact is necessary for transmissionsince wounding prevents signalling (Hick-lin et al., 1973), and Wilby and Webster(1970a) have implicated tight junctions.The idea that communication in develop-ment is via specialized junctions is quitewidespread (Furshpan and Potter, 1968;Wolpert, 1971), and such a mechanismwould permit substances to move from cell-to-cell but not into the extracellularmedium.

A distinction has been drawn betweenthose models which rely on diffusion ofa substance from the boundary region andthose that involve propagated signals(Crick, 1971). In the model given herethe positional information is provided bya sink at one boundary and a source atthe other, the substance diffusing betweenthem. This can be contrasted with thephase shift model (Goodwin and Cohen,1969) in which the boundary acts as apacemaker and positional information isprovided by two signals propagated fromthis region. It is, of course, possible tothink of numerous variations of thesemodels (Cohen, 1971), but the distinctionbetween propagated signalling and diffu-sion remains. Crick (1970, 1971) has em-phasized the importance of considering

TABLE 4. Regenerating 12 onto 12 . . . F : formation of structures at the junction.

Regeneration time(hours)

579

121518

combinations

109

10192321

Normal

1 (10%)3(33%)2 (20%)7 (30%)

19 (82%)18(86%)

Heads

2(20%)1(11%)2 (20%)3(16%)2 (9%)3(14%)

Results

Heads and Feet

4(40%)1(11%)1 (10%,).-5(16%)00

Feet

3 (30%)4 (44%)5 (50%)0(32%)2 (0%)0

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656 WoLPERT, HORNBRUCH, AND CLARKE

mechanisms based on diffusion and hasshown that it is possible to set up a gradi-ent over distances of about 1 mm withinseveral hours. For signalling, one mighttake aggregation of the cellular slime moldsas a model since there is good evidencethat this involves propagation of a periodicsignal (Cohen and Robertson, 1971). Theperiod is about 5 min and it propagatesat about 1 /Am/sec.

The recent work of Wilby and Webster(1970b) has shown that one head end can

inhibit another head end forming overquite long distances and that this inhibi-tion can be propagated in a proximo-distal direction. We have used this systemto investigate the time required by thegrafted head to establish inhibition usingdifferent lengths of hydra.

The plan of the experiment using H.attenuata is shown in Figure 8. After vari-ous times the host head is removed to give1231H and the animals observed to see ifa new head regenerates from the 1 region.The absence of a head is taken to mean

! > Grafted head

> Inhibited

*> not inhibitedRegenerated head

FIG. 8. Plan of experimental scheme to test timetor grafting with distance. The host head is re-moved at 0 time. On the right hand side thegrafted head is placed in position after removal ofthe host head, while on the left hand side it isplaced before removal of the host head.

that the grafted head has inhibited headformation at the 1. Figure 9 shows the per-centages in which head regeneration wasinhibited as a function of grafting time.The grafting times for 50% inhibition for123/H, 12/H, and 1/H are .respectively—8 hr, —2 hr, and -f-6 hr at 18 C.

These results show clearly that the timerequired for a grafted head to be able toinhibit the development of another headregion at some distance from it is verysignificantly altei-ed by this distance. Itmust be grafted on 8 hr before host headremoval for a 123 but can be grafted on6 hr after host head removal for a 1 (thatis, 14 hr later than for the previous case).This dependence on distance, with a timescale of hours, would seem to excludea comparatively fast periodic signallingmechanism of the type observed in slimemolds.

The values from Figure 9 do not givesignal transmission time directly but onlythe times when the head must be grafted.As pointed out above, it is still possibleby grafting to prevent the regenerating endfrom forming a head, the / region becom-ing determined only after a period of hours.We found this time to be 13 hr by graftinga head onto the regenerating 1 region atvarious times and scoring for the presenceof a second set of tentacles, which wastaken as evidence for new head end deter-mination. The total signalling time can beestimated as being that between graftingand determination, i.e., 21, 15, and 7 hrfor lengths 123, 12, and 1 respectively.

In order to see if these experimentalresults are consistent with diffusion wehave simulated the experiments on a com-puter, by assuming for simplicity that ahead will not form from the end of the/ region provided that the concentrationof the morphogen at that point is not be-low its initial value at or after 13 hr. Forthe simulation we used a hypotheticalhydra slightly different from that de-scribed above, but as the results are com-parative the differences are not important.The results are given in Table 5. It shouldbe noted that the diffusion constant is

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100

80

60

I

20

A\A

X = 123/H

o = 12/H

, / H

-12 -8 -4 0hours

FIG. 9. Results of experiment on % inhibition forvarious grafting times according to Figure 8. Each

determined by finding that value whichmakes the time of grafting of the 123jHcorrespond to the observed time. Thevalues of 12/H and 1/H are then deter-mined using this diffusion constant. Theexperiments were repeated at 26 C andthe simulation again carried out. In viewof the large reduction in determinationtime, it was encouraging both that thesimulation gave very reasonable resultsand that the diffusion coefficient was onlyslightly increased, as would be expectedtheoretically.

The experimental results showed for thefirst time quantitative data on the varia-tions of signalling time with distance in apositional field. They show that the timesincrease considerably with the distance

4 8 12 15

point represents 10 grafts.

18

over which effects are transmitted andseem to exclude any mechanism based onrapidly propagated periodicities as sug-gested in early versions of the phase shiftmodel. The speed of signalling at 18 C isslow, being about 100 ^m/hr or 2 celllengths/hr. This is very slow comparedeven with cytoplasmic movements whichare about 10 Mm/sec (Wolpert, 1965). Theexperimental results suggest a roughlylinear relationship between transmissiontime and distance which would be consis-tent with a slowly propagated signal, butwould also not be inconsistent with diffu-sion. Superficial arguments based on timefor diffusion being proportional to lengthsquared can really only be applied tochanges of overall scale between otherwise

TABLE 5 Theoretical and experimental parameters for the establishment of inhibition.

TemperatureHead determination times

(hours)Diffusion constant (cm2/sec)Grafting times (hours)

123/H12/H1/H

18C13

2.0 x 10-7

Experiments Theory- 8 - 8- 2 - 2+ 6 +3.2

28 C2

2.2 X 10-'Experiments Theory

- 9 - 9- 6 -4.7- 2 -1.5

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identical situations. Using a very simplemodel based on a diffusible morphogenit has been found that simple intuitiveassumptions about the relationship be-tween time and distance cannot be madewhen different grafts are used, since overquite a large range of distances an approxi-mately linear relationship holds. The diffu-sion simulation fits the observed datarather well, and the correspondence be-tween our value of the diffusion constant2.00 X 10-7 c m 2 / s e c a n d crick's (1970)estimated value of 2.3 X 10~7 cm2/sec isremarkable. However, in view of the vari-ability in the experimental data and thecrudeness of our model, this should be tak-en as no more than making diffusion aplausible mechanism.

DYNAMICS OF REGIONS AWAY FROM

THE BOUNDARY

The experiments on the dynamics ofhead end formation were concerned witha region at the new head end boundary.A plausible interpretation of those changescould be given in terms of changes inS and P. How does P change away fromthe boundary? For example, while region1 is becoming H, is region 2 becomingregion 11 The available evidence is thatchanges in P away from the boundary areslow, particularly when P is increasing.

In order to study changes in P awayfrom the boundary we have developedan assay for region 1 based on Webster's(1966a) observation that if region 1 isgrafted into the gastric region of an intacthost it is absorbed, but if the host's headis removed a new axis will be induced.Using this assay we have investigated thetime for region 3 to become a region 1in three different situations: (i) a regen-erating 34 . . . F, (ii) a regenerating23 . . . F, (iii) H/3 . . . F. Only in (i) isthe 3 at the boundary; in the other twocases it is away from the boundary. Whilethe time for (i) was about 9 hr, the timesfor the other two cases were 48 hr orlonger. This was a very unexpected ob-servation and has very important conse-quences and implications. It shows that in

a regenerating animal, the relatively rapidchanges in P are localized to the boundaryregion as indicated in the model and thatchanges in the value of P away from theboundary are very slow. It also confirms inan elegant manner that a simple modelbased on a diffusible substance is not ten-able since such a model would predictrapid changes in P in the 3 of a H/34 . . . F.

There is some evidence that P can de-crease more rapidly than increase awayfrom the boundary. If the if of a H12/12 . . . F is removed at various times aftergrafting, one can determine when struc-tures no longer form at the junction. Thistime is about 8 hr and is presumably thetime required for the P value of the proxi-mal 12 to have decreased to that equiva-lent to a 34.

The analysis and interpretation of theabove results are complicated by the ab-sence of a cut surface as well as the pres-ence of the bud. Nevertheless it is possibletentatively to propose a model to accountfor the changes in P away from the boun-dary. The simplest models would seem tobe based on slow diffusion of P or ona follow-up servomechanism (Wolpert,1971), P being synthesized when it is lessthan S and broken down when it is morethan 5. We cannot exclude that the slowchanges in P away from the boundary maybe due to a requirement for cell divisionas in the insect epidermis (Lawrence et al.,1972). This is clearly an area that requiresintensive investigation.

CHANGES IN POLARITY

The problem of the basis of polarity isa very old and fundamental one (seeWolpert, 1971). In hydra polarity is mani-fested by heads forming at a distal cutsurface and feet at a proximal cut surface.It would be a significant advance if thebasis of this polarity were known. A dis-tinction can be drawn between two differ-ent, but not mutually exclusive, ways inwhich polarity of the system could bedetermined: one based on global proper-ties and the other on local structure (Wol-

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pert, 1968). In a global mechanism polar-ity is a property of the system as a wholeand could be determined by the slope ofa gradient in some substance as suggestedby Lawrence et al. (1972); the individualcells would not be polarized but wouldrespond to the system gradient. This canbe contrasted with a local structural mech-anism in which each cell is polarized: itcould, for example, have a polar transportsystem.

The model we have presented suggeststhat polarity is a global property and thatpolarity is determined by the interactionbetween the two gradients. If a piece ofhydra gastric region is isolated, at themoment of isolation the differences in theS and P values at the distal and proximalsurface may be quite small but diffusionof S will result in S decreasing at the distalend while at the proximal end it willincrease. This obviously amplifies the dif-ferences and provides a simple mechanismfor preventing, for example, head forma-tion at both ends.

When symmetrical pieces of hydra aregrafted together with opposite polarityeach behaves like a separate positionalfield. For example, F . . . 1 /I . . . F formsone or two heads in the middle. This iswhat is to be expected in terms of themodel since the concentration of S will belittle perturbed by such grafts. This typeof graft can be contrasted with the behaviorof grafts of the type 3/12 . . . F and 56/12 . . . F. In all combinations of 56/12 . . . Fthe distal region forms a foot suggestingthat the polarity of region 56 has beenreversed. Grafts of 3/12 . . . F also givefeet at the distal end of the 3 in a signifi-cant number of cases. We cannot offer afull explanation as to why a foot shouldform at the distal end in such grafts, butwe can account for the inhibition of headformation there. This can be seen, forexample, from the distribution of 5 fol-lowing the formation of 56/12 . . . F. Sincreases rapidly at the 5 region and pre-vents head formation there.

Reversal of polarity can also occur withgrafts of 123/1, the proximal 1 forming a

head at the cut surface and a foot formingat the junction, even though the pieces arejoined with normal polarity. This maybe accounted for by leakage of S from thecut surface.

A most important set of experiments onpolarity has been carried out by Wilby andWebster (1970a,fo). By grafting a headonto the proximal region of a H123 andthen removing the host head to give aH/321 they showed that head regenera-tion from the 1 could be inhibited. Thisshowed that a signal from the head couldbe transmitted in a direction opposite tothe normal direction. This experiment hasbeen exploited, as described above, to in-vestigate time/distance relations for thetransmission of the signal. An even moreimportant finding was that the signallingfrom the grafted head was in no way re-lated to the regeneration polarity of the123 and that the time course of this changein polarity was very slow, taking up to72 hr. From their studies they concludedthat inhibition of head formation wasquite independent of the reversal of polar-ity and required some other factor suchas reversal of active transport. This waspartly based on their find that removal ofthe grafted head at various times duringpolarity reversal resulted, in a significantnumber of cases, in a head forming notat the ends but near the middle, a resultthey regard as being incompatible withmechanisms based on diffusion.

We believe that their conclusions are notjustified and that their results do not ex-clude a model of the type we have pro-posed in which there is only one diffusiblegradient which determines the value of themore stable second gradient. For example,the very long time required to reversepolarity is quite consistent with our ob-servation that regions away from theboundary increase their P very slowly. Toreverse polarity the 3 in H/321 must in-crease and, as we have shown, the P of a3 in H/34 . . . F increases very slowly.The mechanism whereby the P of 1 fallsis not clear.

Support for the idea that tissue polarity

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is primarily due to a graded distributionof cell types or cellular constituents andnot due to the polarization of individualcells comes from the elegant studies ofGierer et al. (1972). Using their techniqueof reconstructing hydra from disaggregatedcells they showed that cells derived frommore distal regions consistently formedthe head regions when combined withcells from more proximal regions. Theyalso showed that the property determiningpolarity is rather stable and is not lostduring disaggregation.

THE EFFECT OF CHEMICAL AGENTS

The grafting experiments have enableda model to be put forward in terms ofgradients. Even though controversy re-mains as to how the gradients interact andare maintained, the model we have pro-posed can account for a wide variety ofresults. What is urgently needed, but verydifficult to obtain, is some clue as to thebiochemical basis of these gradients. It israther depressing that no rigorous bio-chemical correlate of gradients in hydra orany other system has been obtained.Clearly, crucial changes occur within thefirst few hours of regeneration. Preliminaryinvestigation by Clarkson (1969a,£>) sug-gested that this was accompanied by anincrease in RNA synthesis.

Much of the difficulty in identifying Pand S lies in the absence of a suitableassay. If, as we have suggested, S is a dif-fusible substance, passing from cell-to-cellacross specialized junctions, and which isnot able to get out of the cell, then isolat-ing substances from macerated hydra andapplying them is hardly promising. Evenif one could get the substance into the cells,it is worth considering what one mightexpect. In terms of the current model,increasing 5 everywhere along the hydrawould in the short term (several hours)simply block head formation. Much longerapplication (48 to 72 hr) would be re-quired to cause P to rise in the proximalregion of, for example, isolated digestivezones, so that a head would form there. An

examination of the model suggests that itis quite hard to alter polarity. The modeldoes however suggest that interference withsynthesis of S in regenerating animals couldgive an enlarged head end with possiblyadditional tentacles. It also suggests thatinhibition of synthesis of P will inhibithead regeneration. It is important to re-member that in its present form the modeldoes not include foot end formation andthe possible influence of the foot on thehead end. This is very relevant since asignificant feature of treatment with avariety of agents is that a foot can format a regenerating head end.

Feet form at a regenerating head endwhen hydra are treated with dithiothreitol(Hicklin et al., 1969), colchicine and lowtemperature (CorfE and Burnett, 1969,1970), different species responding differ-ently to such treatment. While it wastempting from such results to implicatemicrotubules (Wolpert et al., 1971), weconsidered it necessary to test a greaterrange of compounds to determine the spec-ificity of such effects, since distal regenerat-ing ends induced proximal structures whentreated with actinomycin D (Clarkson,19696). We have, therefore, subjected in-tact hydra, hydra from which the head hadbeen removed and a wound made in thegastric region, and isolated gastric regionsto short (4 hr) and long (24 hr or more)term exposure to a wide variety of chemi-cal insult at 26 C. Two species were used,H. litloralis and H. aitenuata. The ap-proach was essentially a screening process;the approximate lethal concentration wasfound and the hydra then treated withslightly lower concentrations. The resultsare shown in Table 6 which is only meantto provide a general summary of the re-sults. The concentrations, for example, aresometimes averages for the different species.The results showed that varied and appar-ently unrelated compounds could inhibitregeneration of a head end, as judged bythe appearance of tentacles in H. littoralis,and that these compounds could also bringabout foot formation at the head end. Thisalmost never occurred with H. attenuata.On the other hand, several compounds

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TABLE G. Results of screening a variety of chemical compounds for their effect on hydraregeneration.

Compound

Theophyllinei\a-O=-DibutyrylAdenosine cyclicAMPSodium amytal2,4-DinitrophenolOligomycinAtractylosideValinomycinRotenoneSodium fluorideCarbonyl cyanide2,6-dichlorophenol-

indophenol2-Deoxy-D-glucosePhenazine

methosulphateEthidium bromideNicotinamideRutanolPhenyl ethanolDiamideOuabainConcanavalin ATrypsinEGTA8-chloroxantheneSodium lauryl

sulphatelodosobenzoic acidOncovin5-methyl-cytosineMethyl glyoxal

Bis (guanylhydrazone)Thioridazine HC1Cyproheptadine HC12-heptyl-4-hydroxy-

quinoline-Noxide

Concentration

10"= M

IC-'M3 x 10-' M") X 10"= M10 j ig /ml10-' M

10"= M10"' M5 X 1 0 " ' M10-7

M

10"= M

10-!M10"GM

4 x 10-= M10-' M0.1%0.1%10-4 M10"= M200 / ig /ml0.01%10-' M0 . 1 %10"' M

10"= M2 x 10-'M4 x 10"3M10-' M

10-° M10-" M10- 'M

Time(hours)

48

"\2424242424242424

2424

962424

42424

44

2444

2444

24

184824

& 24

& 24

& 24

& 24

Inhibition

j L

+ ++ +

+ + ++ ++ + +

+ + ++

+

+ + + +

++ + ++++

++ + ++

+ +++ +

+ + ++ + ++ + +

Polarityreversal

-1. 4-

+++ + + ++++ + +

+ ++

++ +

++

+ + +

+ *+ +

+ + ++ »

Extratentacles

+

+ +

+ +

+ +

+ +

Structuresat a

wound

+ +

+

+

+ ++ +

+

++ +

1 These are the two cases where a foot formed at the distal end of a H. attenuata.

FIG. 10. Photograph of a hydra treated with oligo-mycin for 24 hr, after 8 days, showing a foot atthe original head end to the left. Buds whichhave not detached cluster about the foot end.

could lead to extra tentacles forming inH. attenuata; this was never observed withH. littoralis. The single most dramatic re-sult was obtained with oligomycin on H.littoralis. Treatment of intact animals for24 hr results in withdrawal of the tentaclesand the formation of a foot at the distalend (Fig. 10). Since oligomycin is a mito-chondrial poison one can begin to thinkof mechanisms in which ATP productionor level is an important feature of thegradients. Inhibition of energy productioncould explain the action of the otheragents. This is now under investigation.Until more progress is made in this direc-

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662 WOLPERT, HORN'BRUCH, AND CLARKE

tion care must be taken in interpretingthese results. It must be remembered thatthe results in Table 5 represent only aninitial screening, and more detailed ob-servations need to be made with com-pounds such as concanavalin A. The re-sults do emphasize that it is necessary tobe particularly cautious in interpretingexperiments in which claims are made forspecific substances inducing extra tentaclesin H. attenuata (Schaller, 1973) or feet inH. littoralis.

If positional information is universal ashas been suggested, then it is likely to beassociated with some fundamental cellularactivity such as energy production and isunlikely to involve some obscure molecularspecies. Cyclic AMP is an obvious candi-date, but our preliminary results are notencouraging. Hydra contain about 0.1 ngand the relative concentrations H12: 34B:56F: are 3.0: 1.3: 3.5. Finding the bio-chemical basis of the gradients is going totake inspiration, hard work, and luck. Thebiological data and model presented heredo at least provide an idea of what to lookfor and, thus, recognize the biochemicalsystems when they are found.

REFERENCES

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Positional Information and Positional Signalling in Hydra