/ . Embryol. exp. Morph. Vol. 28, 1, pp. 209-222,1972 2 0 9

Printed in Great Britain

The uptake and transfer of radioactivecompounds in axial grafts of Hydra

By J.A.OLSON1

From the Department of Biology, as Applied to Medicine,The Middlesex Hospital Medical School

SUMMARY

Hydra littoralis and H. attenuata were labelled with 45Ca, 32PO4, or [3H]thymidine,either by exposing them to a medium containing the isotope or by injecting the isotope intotheir gut cavities. Various portions of labelled and unlabelled hydra were grafted togetherin diverse combinations, and the rate of transfer of radioactivity to the unlabelled portionwas assessed. The relative transfer rates of the isotopes were 32PO4 >

45Ca2+ > [3H]thymi-dine. The transfer rates for 32P-labelled compounds and 45Ca were unaffected either bythe polarity gradient (from head to foot and vice versa) or by the existence of potentiallyseparate biological fields. Isotopes were not readily transferred between hydra or pieces ofhydra which were in apposition but not grafted together. A rapid and sensitive method forthe measurement of hydra mass is also described.

INTRODUCTION

Hydra have a remarkable ability to regenerate their initial forms, essentiallyby morphallactic transformation. Since each part of a hydra regenerates in amanner characteristic of its initial position in the intact organism (Wolpert,Hicklin & Hornbruch, 1971), this rapid, ordered, and consistent re-establish-ment of biological pattern, and indeed the development of the specific biologicalform initially, must necessarily be denned by some type of intrinsic coordinatesystem with specific boundary values (Wolpert, 1969, 1971). Suggested mechan-isms of pattern regulation fall into two major classes: (1) positional informationor continuous gradient models (Wolpert, 1969; Goodwin & Cohen, 1969); and(2) cascade or induction type models, which involve the sequential action of mor-phogenic factors (Mercer, 1964; Rose, 1952; Webster, 1971). With the exceptionof the phase shift concept of Goodwin & Cohen (1969), the localized synthesis ofone or more signalling substances, which are then transmitted in a controlledway throughout a biological field, is an essential aspect of all models.

The chemical nature of such morphogenic or position determining substances,if indeed they exist, and the mechanism by which signalling occurs among cellsin a biological field, however, is largely unknown. Quite possibly, information

1 Author's address and affiliation: Department of Biochemistry, Faculty of Science, MahidolUniversity, Rama VI Road, Bangkok, Thailand. Staff member, the Rockefeller Foundation.

14 E M B 28

210 J. A. OLSON

may be transmitted through low resistance junctions formed between adjacentcells (Furshpan & Potter, 1968; Lowenstein & Penn 1967; Pitts, 1971).

In order to elucidate the mechanism of signalling, some prior understandingof the transfer of compounds among cells in normal and regenerating hydrawould seem to be essential. I have consequently labelled hydra with radioactivecalcium, phosphorus and thymidine, and then have measured the transfer ofradioactivity from labelled to unlabelled portions in various kinds of axialgrafts.

MATERIALS AND METHODS

Maintenance of hydra colonies. Hydra attenuata, kindly sent us by Dr CharlesDavid from Tubingen, and H. littoralis were grown in covered plastic dishesat 20-22 °C in a medium containing 10~3 M-NaHCO3, 10"3 M-CaCl2, 10~3 MTris (hydroxymethyl)aminomethane, 10~3

M-KCI and 10~4 M-MgCl2 at pH 7-5-7-8 (Muscatine, 1961). Hydra were fed with newly hatched nauplii of Artemiasalina, obtained from California Brine Shrimp Inc., 711 Hamilton Avenue,Menlo Park, California, U.S.A. and were cleaned daily after feeding.

Determination of hydra mass. Hydra vary greatly in size, with newly detachedbuds often being one-fifth or less the size of mature animals with two or morebuds attached. A simple, rapid and sensitive method for determining the mass ofhydra was therefore developed.

A hydra, or as little as one-tenth of a hydra, was sonorated in 0-4 ml distilledwater for 20 s at room temperature with a Dawe Soniprobe, Type 1130 A,18-22 Kc/s (kHz), equipped with a \ in. (3-2 mm) titanium micro tip (DaweInstrument Limited, Acton, London, W. 3). A current of 0-8 A (setting no. 2) wassufficient to disrupt the hydra within about 10 s to a stable turbid solution, whoseoptical density was unaffected by sonorating for an additional 60 s. A spectrumof the disrupted hydra is given in Fig. 1. Two effects are superimposed in thisspectrum, the light scattering properties of the sonorated particles of hydra andthe absorbancy of proteins and nucleic acids in the near ultraviolet region. Aplateau in optical density exists around 250 nm, and the mass of the sonoratedhydra was found to be directly proportional to the optical density at that wave-length, as shown in Fig. 2. On the average, one H. littoralis gave an optical den-sity (O.D.) of 0-5, and one H. attenuata an optical density of 1-0. The range ofoptical densities for a single H. littoralis, from newly detached buds to matureanimals with several attached buds, was 0-15-0-85. The average optical densityper animal, of course, is greatly affected by the state of nutrition, parasitic andbacterial contaminants, and other factors influencing the colony.

In order to determine the relationship between the optical density at 250 nmand the wet and dry weights of hydra, 40 randomly selected H. littoralis werefasted overnight, cleaned and then filtered on a tared 1 cm square of nylonmesh no. 300. The nylon square was quickly blotted on a piece of laboratorytissue and immediately weighed. Thereafter the hydra were resuspended in

Axial grafts of hydra 211

1-6

1-2

0-8

0-4

250 300Wavelength (nm)

350

Fig. 1. The spectrum of a sonorated suspension of two fasted H. Httoralis,with buds, in 0-4 ml H2O.

10 ml of water and sonorated for 20 s at setting no. 4. The average wet weightper hydra was found to be 0-23 mg, or equivalent to a volume of about 0-23 jul,and the wet weight per optical density unit at 250 nm was calculated to be1-46 mg, or 1 -46 ja\. The results of several experiments agreed within 5 %.

In separate experiments 40 randomly selected H. Httoralis were weighed ona tared nylon square, transferred to normal medium, and filtered on a taredMillipore disc (0-3 jam pore diameter), which was then dried at 70 °C in anoven to constant weight. The average dry weight to wet weight ratio was 0-13with a variation of + 2 %.

Isotopes and counting procedures. 45CaCl2, with a specific activity of 13-8 mCi/fig calcium, H3

32PO4 with the specific activity of 108 fiC\j/ig phosphorus, andthymidine (methyPH), with a specific activity of 5 mCi//*mole were obtainedfrom the Radiochemical Centre, Amersham. When desired or necessary, thestock solutions were diluted with the corresponding nonradioactive compoundor were neutralized with Tris (hydroxymethyl) amino methane buffer toapproximately pH 7-5. Isotope solutions were frozen and stored at - 2 0 °C.

Radioactive aqueous samples (0-4 ml) were added to 4-6 ml of a liquid scin-tillator consisting of 4g 2,5-diphenyloxazole (PPO), 0-2 g dimethyl l,4-di-2-(5-phenyloxazole)-benzene (dimethyl POPOP), 60 g naphthalene, 20 ml ethyleneglycol and 100 ml methanol in dioxane made up to 11. (Bray, 1960). Optimal gainsettings for 32P, 45Ca, and 3H were 3, 30 and 60 % respectively. At a concentra-tion of 20 % water, 32P quenched less than 2 %, 45Ca less than 5 %, and tritium

14-2

212 J. A. OLSON

0-8

c 0-6

0-4

0-2

1 2 3Hydra/0-4 ml

Fig. 2. Absorbance of various dilutions in H2O of a sonorated suspensionof 25 fasted H. littoralis in 1 ml H2O.

about 65 % under standard counting conditions in a Tricarb liquid scintillationcounter. Net cpm were corrected for the half life of 32P (14-3 days) and 45Ca(165 days) when necessary.

General labelling and grafting procedures. Usually 10-25 medium-sized hydrawith one or no buds were cleaned of attached mucus and other debris, fed, re-washed and then suspended in normal medium. In some cases a given isotopewas added to normal medium, and the hydra were left for 1-2 days at 20-22 °Cwith daily changes of the medium. In other cases the isotope was injected into thegut cavity of the fed hydra with a Hamilton hypodermic microsyringe equippedwith a fine drawn-out polyethylene tip (Campbell, 1965), and the hydra wereincubated for 0-5-2 days at 20-22 °C in normal medium, also changed each day,which contained 10~3 to 10~4M of the appropriate nonradioactive compound(phosphate or thymidine).

After incubation labelled hydra were washed, and several animals wereimmediately sonorated and counted. Under a dissecting microscope, otherlabelled animals were cut transversely with a razor-blade fragment on a blackplasticine surface under normal medium, and the labelled pieces were strungaxially on a thin blond hair together with non-radioactive segments of hydra.

Axial grafts of hydra 213

Fig. 3. Designation of regions of the hydra, where H indicates the head region,including tentacles; B, the budding region; and F the foot. The figure of the ex-tended unfed H. Httoralis is traced from a photograph, taken by Miss AmataHornbruch.

After removal of the hair (^ 1 h), grafts were incubated at 26 °C. Transverselevels of the hydra were designated by the symbols given in Fig. 3, and graftingcombinations are expressed in the standard way (Wolpert, 1969; Wolpert et ah1971). Initially labelled sections in grafts are designated with an asterisk, e.g.H12*/34B56F means that a radioactive H12 portion is grafted to an unlabelled34B56F.

At various times thereafter, the grafted hydra were cut into a suitable numberof transverse sections, which were individually washed in normal media andplaced in 0-4 ml H2O. Each section was sonorated, the optical density at 250 nmwas determined in 0-5 ml microcells in a Unicam SP 800 spectrophotometer,and the whole solution was added to 4-6 ml Bray's scintillation fluid and countedfor 10 min, or longer if necessary.

214 J. A. OLSON

Table 1. Transfer of radioactivity from sections labelled with tritiatedthymidine to unlabelled sections in grafts of H. attenuata

R values of initially unlabelled sections

Hours

il2345

2327

H12*/34B56F

34B

000-53000-551-44-7

56F

00000000

H1234B/56F*

H12

00000000

A

34B

000-620140001-5

The specific activity of a section is expressed as counts per minute (cpm) peroptical density unit for a 0-4 ml volume, and the relative specific activity (R)in different parts of the hydra is expressed as a percentage of the specific activityof the most radioactive part, i.e.

R = c p m / o . D . ncpm/o.D. a

where section a has the highest specific activity in the hydra graft and n denotesany other section.

RESULTS

Transfer of radioactive compounds in grafts labelled with [3H]thymidine.Ten H. attenuata were each injected with about 0-3 /i\. of tritiated thymidine(ca. 1 jLtdl/A.) and left in normal medium containing 10 [ig unlabelled thymidine/ml for 3 days with daily feeding. Thereafter, H12*/34B56F and H1234B/56F*grafts were made, and at various times after grafting, from 20 min to 27 h,animals were cut into three sections, one consisting of the original radioactivepiece, the second the unlabelled piece adjacent to it and the third the unlabelledpiece farthest removed from the labelled section. In all cases the specific activityof the initially labelled section at the time of analysis was between 1000 and3000 cpm per O.D. unit. The results are presented in Table 1.

Quite clearly no significant transfer of radioactivity occurred until 23-27 hafter grafting. Grafting was essential for any transfer to occur, since unlabelledpieces just incubated together with the labelled segments had R < 0-1. Aftersonoration of the initially labelled pieces, over 50 % of the total tritium wasfound in the TCA insoluble precipitate retained by a Millipore filter (porediameter 0-22 /im). In all likelihood, this is a minimal figure for the TCAinsoluble portion, since losses undoubtedly occurred during handling and filtra-tion.

Axial grafts of hydra 215

A. Uptake

Hours8000 -

4000

2000 -

1000

B. Release

20 60 100

HoursFig. 4. The rate of uptake (A, upper curve) and release (B, lower curve) of 45Ca2+

by hydra. A group of 50 fasted H. littoralis was suspended in 2 ml of medium con-taining 5 x 106 cpm in 0-61 /*mole 45Ca2+. After 48 h the labelled hydra were placedin normal non-radioactive medium. At various intervals during both the uptakeand release phases, 2-3 hydra were removed and analysed, and cpm/o.D. valueswere calculated. Since 45Ca2+ uptake will be proportional to the difference betweenthe [45Ca] in the medium and that in the hydra at any given time, Co is expressed ascpm 45Ca per O.D. unit, or l-46xcpm 45Ca//tl. Concentration values are graphedlogarithmically and time linearly.

Transfer of ^Ca from labelled to unlabelled sections in grafts of H. littoralis.Before studying the transfer of 45Ca in grafts, the rates at which radioactivecalcium was taken up and released by hydra were assessed. As shown in Fig. 4(upper), radioactive calcium in the medium exchanged with hydra Ca2+ atan exponential rate with a half life of about 2 h. The release of 45Ca2+ fromhydra, on the other hand, seemed to occur in two distinct steps, a rapid exchangeof \-\ the calcium with a ^ of about 6 h, and a slow exchange with a t$ of about75 h (Fig. 4, lower). The above experiments were conducted with approximatelythe same concentrations (10~3-10~4M) of calcium in the hydra and in the medium.When much lower concentrations of Ca2+ were present in the medium (10~5-10~6 M), hydra tended to maintain their internal Ca2+ concentration (Fig. 5).

216 J. A. OLSON

60

~_ 40U

u

20

10" 10 10 4

[Ca2 + ] in medium10 10 2

Fig. 5. The steady-state ratio of Ca2+ in hydra to Ca2+ in the medium at variousinitial Ca2+ in the medium. Eight groups of about 15 cleaned H. littoralis wereeach suspended in 2 ml normal medium except that the initial CaCl2 concentrationvaried from 3 x 10~G M to 0 0 5 M. All solutions contained 25xl06cpm 45Ca perml. At 18 h (O), 47 h (Q) and 114 h (A), samples of 2-3 hydra were washed andanalysed. On the basis of cpm/o.D. values, calcium concentrations were calculated.Hydra tolerated 001 M-CaCl2, but not higher concentrations. 45Ca values below10~5 M are somewhat unreliable because of ion leakage from hydra.

Various parts of the hydra, labelled with 45Ca as described above, had essentiallythe same specific activity, ± 10 %, in the steady state.

Since an appreciable portion of 45Ca was retained in the hydra for relativelylong periods, groups of about 50 H. littoralis were labelled by being incubatedin 2 ml of medium containing 4x 10~6 M-45CaCl2 with 5x 106cpm for 48 h,and grafts were made. The transfer of labelled calcium, expressed as R values inH1*/2...F and H...B/56F* grafts is given'in Table 2. Clearly 45Ca moved muchmore rapidly than did thymidine. Transfer did not occur to any appreciabledegree, however, by release from the labelled section into the medium followedby uptake into the unlabelled section. Labelled and unlabelled pieces which werein apposition, but not grafted together, for example, had R values of less than0-1 even at 44 h. Furthermore, unlabelled hydra placed together with labelledgrafts took up essentially no radioactivity (R < 0-3). Although 45Ca was lostprogressively from all pieces during incubation, the specific activity of the initi-ally labelled sections of grafts at the time of analysis varied from 4000 to 6000cpm/o.D. unit for short incubations, and from 300 to 1500 cpm/o.D. unit forincubations of 19 h or more.

Axial grafts of hydra 217

Table 2. The transfer of i5Ca2+ from labelled to unlabelled sectionsin grafts o/H. littoralis

R values of initially unlabelled sections

Hours

it21-4 !192244

t

23

1-85-3—5-39-7

At I h the

H1*/234B56F

4B

0-80-51-5—3 03-7

total unlabelled

56F

0-10-8—00-6

section of hydra was

HI

002-64-43 0

H1234B/56F*A

23

2-400

12-42-3

11-5

4B

0-92-2

12-48-2

300

analysed as a single piece.

Transfer of ^P-labelled compounds from initially labelled to unlabelled sectionsin grafts of hydra. When H. littoralis was incubated in normal medium con-taining labelled inorganic phosphate, the 32P uptake into hydra was initiallyrapid and then plateaued between 1 and 2 days. Each major section of hydraequilibrated in this way had the same specific activity, ± 12 %. The 56Fsection was always somewhat more strongly labelled than sections H and12 (0-01 < P < 0-05). Upon placing these 32P-labelled hydra in normal mediumcontaining nonradioactive inorganic phosphate, approximately one-third of theradioactivity was released within 12-24 h, but thereafter very little radioactivitywas lost during a subsequent 4-day period. For grafting experiments about 10 H.attenuata were injected with approximately 0-2 /A. of 32PO4 solution which con-tained 2 fiC\ and 20 pg PO4 per /il. The hydra were incubated in normal mediumcontaining 10~3 M phosphate overnight and then were grafted in the conventionalway.

As shown in Table 3, significant radioactivity was transferred to the most dis-tant slice within one hour of grafting. The R values steadily increased with timeand by 46 h all sections had R values of 49 or higher. As in other cases, R valuesfor grafts which did not hold together, or for pieces of unlabelled hydra whichwere incubated together with highly labelled hydra, were 0-4 or less during5 h of incubation. In all experiments with 32PO4, the initially labelled piece hada specific activity of 5000-20 000 cpm/o.D. unit at the time of analysis. Similarexperiments with H. littoralis, in which labelling was achieved by soaking in asolution containing radioactive phosphate for 1-2 days, gave essentially identicalresults.

When labelled sections were sonorated, treated with trichloroacetic acid andfiltered on Millipore filters, over 60 % of the total 32P was present in the acidinsoluble precipitate. The properties of the labelled material in initially un-labelled sections of grafts were not determined.

218 J. A. OLSON

Table 3. The transfer of radioactivity from 32PO^-labelled to unlabelledsections in grafts of H. attenuata

R values of initially unlabelled sections

H12*/34B56F H1234B/56F*

Hours

12345

46

34B

0-63-88-18-6

13-411-773-0

56F

0070-71-31-17-33-5

490

H12

0 10-6103-81-52-9

570

34B

0-53 07-5

17-411-015-378-0

Transfer of *bCa and 32P phosphate in other types of grafts: Pieces of hydralabelled either with 45Ca or with 32PO4 were grafted in various combinationswith unlabelled sections, and the grafts were incubated for 20 h. As shown inTable 4, the transfer of 32P-labelled compounds from labelled to unlabelledsections was roughly the same, regardless of the nature of the graft. Althoughshowing greater variability, 45Ca was also transferred well from labelled tounlabelled pieces in all grafting combinations.

DISCUSSION

In order to calculate approximate specific activities in hydra sections, it wasuseful to develop a rapid, sensitive and reproducible method for determiningthe cell mass of hydra. Sonoration of whole hydra, or parts of it, in a smallvolume for as little as 10 s yielded a relatively stable turbid solution, whoseoptical density at 250 nm was found to be proportional to the number of hydrapresent. Since one would expect hydra to be uniformly labelled when either32PO4 or 45Ca is employed, the observed constancy of the specific activity, ex-pressed as cpm per O.D. unit, in different parts of the hydra, suggests that theprocedure does indeed give a practical measure of hydra mass. Other methodswhich have been used for expressing the cell mass of hydra are total nitrogen,DNA or protein values (Clarkson, 1969 a, b). Techniques for determining thesecompounds, however, are more time-consuming and involved than the simpleturbidity-absorption procedure described here. Expressing data in terms of thenumber of hydra is of course quite misleading, since hydra vary greatly in sizedepending on species, nutrition and stage of maturation.

With respect to isotopic measurement, the direct counting by scintillationspectrophotometry of sonorated suspensions of hydra in a scintillation solutionwhich tolerates appreciable quantities of water was rapid and adequately precise.Autoradiography, although useful in localizing isotopic compounds in cells or

Axial grafts of hydra 219

Table 4. Transfer of radioactivity from 45Gz2+- or 32PO ̂ -labelled tounlabelled sections in grafts ofH. littoralis incubated for 20 h

R values of initially unlabelled sections

Craft combinations

H123*/4B56FH123/4B56F*H123*/321HF65B4*/4B56F1234*/43214321*/1234

32P transfer

233823—2926

45Ca transfer

27, 3524,3033, 1024,5320,2713, 18

tissues of hydra (Campbell, 1965), suffers from the disadvantages that water-soluble materials are usually lost, the time lag between experiment and evalua-tion is long, and grain counting is tedious.

The fact that tritiated thymidine, presumably incorporated solely into theDNA of cells, does not move readily from labelled to unlabelled sections wasmore or less expected. Certainly no obvious movements of radioactivity occur-red within 5 h, and a small but significant transfer of thymidine from the labelledto the initially unlabelled section occurred only after 23 h. This transfer of radio-activity is almost certainly due to the migration of cells. Although the mitoticindex of cell division is more or less uniform throughout the hydra (Campbell,1967 a) cells tend to migrate from the 12 region of the neck either down thegastric column or up towards the tentacles (Campbell, 19676). About 8 daysare required for individually marked cells to travel from the neck down into thebudding region. The present finding that 1-4 to 5 % of the [3H]thymidine label-led compounds move from the H12 region into the gut section in one day isroughly in keeping with Campbell's earlier studies with hydra marked withdye (19676). Also the fact that radioactivity moves less readily from the foot upinto the 34B region accords with Campbell's observation that gastric region cellsmigrate predominantly towards the foot.

Calcium was much more mobile. Within 5 h an appreciable movement ofisotope from labelled to unlabelled sections occurred, and within 44 h allsections were significantly labelled. Clearly calcium transfer was much morerapid than cellular movement, and occurred at roughly the same rate from headto foot as in the opposite direction. The initial uptake of 45Ca ( + fy = 2-1 h)and its phase of rapid release ( —1% = 6 h) generally agree with similar studieson other ions. 24Na, 42K and 82Ba, for example, are taken up by Pelmatohydraoligactis with half-lives ( + t£) of roughly 1^-3 h, whereas the t$ for the releaseof 24Na was approximately 5 h in distilled water (Lilly, 1955).

32P-Labelled compounds, however, moved even more rapidly. Within 30 minof grafting the section adjacent to the initially labelled piece was significantlylabelled, and within 1 h the most distant section also contained radioactivity.

220 J. A. OLSON

It is interesting to compare the rate of movement of 32P-labelled compounds withthe time required for biological determination to take place in hydra. Signallinggenerally seems to be rapid, and in some cases determination occurs in about1 h (Wolpert et al. 1971). For example, the time required for a 4 region to be-come determined as a foot is about 4 h in H1234 sections, but is only 1 h in1234 sections. The simplest explanation for this observation is that some in-hibitory signal from the head is transmitted to the 4 region within 1 h, therebyslowing the rate of foot determination (Wolpert et al. 1971). Although I do notsuggest that phosphorylated compounds are necessarily involved in the controlof pattern determination, it is interesting to note that only 32P-labelled com-pounds, of the three different types of isotopic compounds studied here, weretransferred at a rate consistent with biological signalling times.

Polarity had little apparent effect on the transfer of either 45Ca- or 32P-labelledcompounds from labelled to unlabelled sections of hydra. In reassembledhydra, like H12*/34B56F or H1234B/56F*, the rate of movement of radioacti-vity from head to foot and from foot to head is roughly the same. Similarly, inreverse-polarity grafts, such as H123*/321H and in reverse-polarity regeneratinggrafts such as 4321*/1234 the movement of both 45Ca- and 32P-labelled com-pounds was similar to that observed in reassembled hydra. These measurementsare admittedly somewhat crude, and more precise analysis might yet show somepolarity dependence. Furthermore, the movement of specific compounds ofcourse might well be markedly influenced by polarity. It is interesting to note,however, that biological signals also seem to be transmitted readily in bothdirections with respect to a polarity gradient. For example, a certain symmetryof inhibition exists with respect to the head and foot regions, namely, that a headtends to inhibit the formation of a new head proximally whereas a foot tendsto depress formation of a new foot distally (Wolpert et al. 1971). Furthermore,a hypostome transplanted into a digestive zone inhibits the formation of a newhypostome at the distal end in a significant number of animals (Webster, 1971).

In only one other case has the transfer of 32P-labelled compounds in graftsof hydra been studied (Hopper, 1962). Tentacles, H, or H123 sections weregrafted laterally to the 56 region of host Pelmatohydra oligactis, where eitherhost or graft was labelled, and the presence of 32P-labelled compounds wasassessed qualitatively by'autoradiography. Radioactivity was found to movefrom the labelled to the unlabelled portions within 6 h, was extensively dis-tributed in 12 h, and was more or less equalized in 24 h. In all cases the move-ment of radioactivity was unaffected by the polarity of the graft, or by the nature(graft or host) of the initially labelled part. These findings obviously accord wellwith my own more quantitative observations.

Radioactive compounds might be transmitted in hydra by several possibleways: (1) by direct cell to cell transfer, (2) via the intercellular space (the meso-glia), (3) by circulation through the gut cavity, and (4) by interchange with theexternal medium. Of these four possibilities, only interchange with the external

Axial grafts of hydra 221medium has been ruled out experimentally. Circulation through the gut cavityalso seems unlikely, inasmuch as a gradient of radioactivity always existedfrom the initially labelled to the most distant unlabelled section. If compoundswere extensively excreted into the gut cavity, the radioactivity in all initiallyunlabelled sections should be more nearly the same. Further studies on themechanism of transfer would clearly be most welcome.

The chemical nature of the compounds which are transferred has not as yetbeen defined. Calcium, of course, might exist either as a free ion or as innumer-able chelated forms. Tritiated thymidine is presumably incorporated largelyinto DNA, which probably accounts for its slow movement in grafts. When32P is employed, over half of the radioactivity resides in the acid insolublefraction of the initially labelled hydra. Since phosphate in most macromolecularand smaller components turns over rapidly, however, no deductions can bemade about its transfer form.

In other studies various fractions derived from hydra have been shown tohave inhibitory, and occasionally stimulatory, effects on regeneration (Webster,1971). Except for the nematocyst toxin, tetramethylammonium, however, thesecompounds neither have been well characterized chemically, nor have beenshown to possess specific pattern forming functions in hydra. It would be inter-esting, however, if some of these partially characterized morphogenic substanceswere also rapidly transferred in hydra grafts.

This work was partially supported by a grant-in-aid from the Rockefeller Foundation(Ga-BMS 7006) and by the Nuffield Foundation. The author wishes to express his apprecia-tion for the help of Miss Amata Hornbruch in maintaining and grafting hydra and to Pro-fessor Lewis Wolpert for providing facilities and critical encouragement.

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organization of developing systems. / . theor. Biol. 25, 49-107.HOPPER, A. F. (1962). Transfer of radiophosphorus from parent to bud and from graft to

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(Manuscript received 1 January 1972)