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 into their gut cavities. Various portions of labelled and unlabelled hydra were grafted together in diverse combinations, and the rate of transfer of radioactivity to the unlabelled portion was assessed. The relative transfer rates of the isotopes were 32PO4 > 45Ca2+ ⪢ [3H]thymidine. The transfer rates for 32P-labelled compounds and 45Ca were unaffected either by the polarity gradient (from head to foot and vice versa) or by the existence of potentially separate biological fields. Isotopes were not readily transferred between hydra or pieces of hydra which were in apposition but not grafted together. A rapid and sensitive method for the measurement of hydra mass is also described.

Hydra have a remarkable ability to regenerate their initial forms, essentially by morphallactic transformation. Since each part of a hydra regenerates in a manner characteristic of its initial position in the intact organism (Wolpert, Hicklin & Hornbruch, 1971), this rapid, ordered, and consistent re-establishment of biological pattern, and indeed the development of the specific biological form initially, must necessarily be defined by some type of intrinsic coordinate system with specific boundary values (Wolpert, 1969, 1971). Suggested mechanisms of pattern regulation fall into two major classes: (1) positional information or continuous gradient models (Wolpert, 1969; Goodwin & Cohen, 1969); and

(2) cascade or induction type models, which involve the sequential action of morphogenic factors (Mercer, 1964; Rose, 1952; Webster, 1971). With the exception of the phase shift concept of Goodwin & Cohen (1969), the localized synthesis of one or more signalling substances, which are then transmitted in a controlled way 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 cells in a biological field, however, is largely unknown. Quite possibly, information may be transmitted through low resistance junctions formed between adjacent cells (Furshpan & Potter, 1968; Lowenstein & Penn 1967; Pitts, 1971).

In order to elucidate the mechanism of signalling, some prior understanding of the transfer of compounds among cells in normal and regenerating hydra would seem to be essential. I have consequently labelled hydra with radioactive calcium, phosphorus and thymidine, and then have measured the transfer of radioactivity from labelled to unlabelled portions in various kinds of axial grafts.

Maintenance of hydra colonies. Hydra attenuata, kindly sent us by Dr Charles David from Tübingen, and H. littoralis were grown in covered plastic dishes at 20–22 °C in a medium containing 10–3 M-NaHCO3, 10–3 M-CaCl2, 10–3 M Tris (hydroxymethyl)aminomethane, IO–3 M-KCI and 10–4 M-MgCl2 at pH 7·5– 7·8 (Muscatine, 1961). Hydra were fed with newly hatched nauplii of Artemia salina, 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 detached buds often being one-fifth or less the size of mature animals with two or more buds attached. A simple, rapid and sensitive method for determining the mass of hydra was therefore developed.

A hydra, or as little as one-tenth of a hydra, was sonorated in 0·4 ml distilled water 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 (Dawe Instrument Limited, Acton, London, W. 3). A current of 0·8 A (setting no. 2) was sufficient to disrupt the hydra within about 10 s to a stable turbid solution, whose optical density was unaffected by sonorating for an additional 60 s. A spectrum of the disrupted hydra is given in Fig. 1. Two effects are superimposed in this spectrum, the light scattering properties of the sonorated particles of hydra and the absorbancy of proteins and nucleic acids in the near ultraviolet region. A plateau in optical density exists around 250 nm, and the mass of the sonorated hydra was found to be directly proportional to the optical density at that wavelength, as shown in Fig. 2. On the average, one H. littoralis gave an optical density (O.D.) of 0·5, and one H. attenuata an optical density of L0. The range of optical densities for a single H. littoralis, from newly detached buds to mature animals with several attached buds, was 0·15–0·85. The average optical density per animal, of course, is greatly affected by the state of nutrition, parasitic and bacterial contaminants, and other factors influencing the colony.

Fig. 1.

The spectrum of a sonorated suspension of two fasted H. littoralis, with buds, in 0·4 ml H2O.

Fig. 1.

The spectrum of a sonorated suspension of two fasted H. littoralis, with buds, in 0·4 ml H2O.

Fig. 2.

Absorbance of various dilutions in H2O of a sonorated suspension of 25 fasted H. littoralis in 1 ml H2O.

Fig. 2.

Absorbance of various dilutions in H2O of a sonorated suspension of 25 fasted H. littoralis in 1 ml H2O.

In order to determine the relationship between the optical density at 250 nm and the wet and dry weights of hydra, 40 randomly selected H. littoralis were fasted overnight, cleaned and then filtered on a tared 1 cm square of nylon mesh no. 300. The nylon square was quickly blotted on a piece of laboratory tissue and immediately weighed. Thereafter the hydra were resuspended in 10 ml of water and sonorated for 20 s at setting no. 4. The average wet weight per hydra was found to be 0·23 mg, or equivalent to a volume of about 0·23 μ1, and the wet weight per optical density unit at 250 nm was calculated to be 1·46 mg, or 1·46 μ1. The results of several experiments agreed within 5 %.

In separate experiments 40 randomly selected H. littoralis were weighed on a tared nylon square, transferred to normal medium, and filtered on a tared Millipore disc (0·3 μm pore diameter), which was then dried at 70 °C in an oven to constant weight. The average dry weight to wet weight ratio was 0·13 with a variation of ± 2 %.

Isotopes and counting procedures. 45CaCl2, with a specific activity of 13·8 mCi/ μg calcium, H332PO4 with the specific activity of 108 μCi/μg phosphorus, and thymidine (methy–3H), with a specific activity of 5 mCi/μmole were obtained from the Radiochemical Centre, Amersham. When desired or necessary, the stock solutions were diluted with the corresponding nonradioactive compound or were neutralized with Tris (hydroxymethyl) amino methane buffer to approximately pH 7·5. Isotope solutions were frozen and stored at –20 °C.

Radioactive aqueous samples (0·4 ml) were added to 4·6 ml of a liquid scintillator consisting of 4 g 2,5-diphenyloxazole (PPO), 0·2 g dimethyl 1,4-di-2-(5-phenyloxazole)-benzene (dimethyl POPOP), 60 g naphthalene, 20 ml ethylene glycol and 100 ml methanol in dioxane made up to 11. (Bray, 1960). Optimal gain settings for 32P, 45Ca, and 3H were 3, 30 and 60 % respectively. At a concentration of 20 % water, 32P quenched less than 2 %, 45Ca less than 5 %, and tritium about 65 % under standard counting conditions in a Tricarb liquid scintillation counter. 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 hydra with one or no buds were cleaned of attached mucus and other debris, fed, rewashed and then suspended in normal medium. In some cases a given isotope was added to normal medium, and the hydra were left for 1–2 days at 20–22 °C with daily changes of the medium. In other cases the isotope was injected into the gut cavity of the fed hydra with a Hamilton hypodermic microsyringe equipped with a fine drawn-out polyethylene tip (Campbell, 1965), and the hydra were incubated 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 were immediately sonorated and counted. Under a dissecting microscope, other labelled animals were cut transversely with a razor-blade fragment on a black plasticine surface under normal medium, and the labelled pieces were strung axially on a thin blond hair together with non-radioactive segments of hydra.

After removal of the hair ( ⩽ 1 h), grafts were incubated at 26 °C. Transverse levels of the hydra were designated by the symbols given in Fig. 3, and grafting combinations are expressed in the standard way (Wolpert, 1969; Wolpert et al. 1971). Initially labelled sections in grafts are designated with an asterisk, e.g. H12*/34B56F means that a radioactive H12 portion is grafted to an unlabelled 34B56F.

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 extended unfed H. littoralis is traced from a photograph, taken by Miss Amata Hornbruch.

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 extended unfed H. littoralis is traced from a photograph, taken by Miss Amata Hornbruch.

At various times thereafter, the grafted hydra were cut into a suitable number of transverse sections, which were individually washed in normal media and placed in 0·4 ml H2O. Each section was sonorated, the optical density at 250 nm was 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 counted for 10 min, or longer if necessary.

The specific activity of a section is expressed as counts per minute (cpm) per optical 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 activity of the most radioactive part, i.e.
formula
where section a has the highest specific activity in the hydra graft and n denotes any other section.

Transfer of radioactive compounds in grafts labelled with [3H]thymidine. Ten H. attenuata were each injected with about 0·3 μl. of tritiated thymidine (ca. 1 μCi/μl.) and left in normal medium containing 10 μg 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 radioactive piece, the second the unlabelled piece adjacent to it and the third the unlabelled piece farthest removed from the labelled section. In all cases the specific activity of the initially labelled section at the time of analysis was between 1000 and 3000 cpm per O.D. unit. The results are presented in Table 1.

Table 1.

Transfer of radioactivity from sections labelled with tritiated thymidine to unlabelled sections in grafts of H. attenuata

Transfer of radioactivity from sections labelled with tritiated thymidine to unlabelled sections in grafts of H. attenuata
Transfer of radioactivity from sections labelled with tritiated thymidine to unlabelled sections in grafts of H. attenuata

Quite clearly no significant transfer of radioactivity occurred until 23–27 h after grafting. Grafting was essential for any transfer to occur, since unlabelled pieces just incubated together with the labelled segments had R< 0·1. After sonoration of the initially labelled pieces, over 50 % of the total tritium was found in the TCA insoluble precipitate retained by a Millipore filter (pore diameter 0·22 μm). In all likelihood, this is a minimal figure for the TCA insoluble portion, since losses undoubtedly occurred during handling and filtration.

Transfer of45Ca from labelled to unlabelled sections in grafts of H. littoralis. Before studying the transfer of 45Ca in grafts, the rates at which radioactive calcium was taken up and released by hydra were assessed. As shown in Fig. 4 (upper), radioactive calcium in the medium exchanged with hydra Ca2+ at an exponential rate with a half life of about 2 h. The release of 45Ca2+ from hydra, on the other hand, seemed to occur in two distinct steps, a rapid exchange of the calcium with a of about 6 h, and a slow exchange with a of about 75 h (Fig. 4, lower). The above experiments were conducted with approximately the 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).

Fig. 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 containing 5 × 106 cpm in 0·61 μmole 45Ca2+. After 48 h the labelled hydra were placed in normal non-radioactive medium. At various intervals during both the uptake and release phases, 2–3 hydra were removed and analysed, and cpm/o.D. values were calculated. Since 45Ca2+ uptake will be proportional to the difference between the [45Ca] in the medium and that in the hydra at any given time, Co is expressed as cpm 45Ca per O.D. unit, or l·46×cpm 45Ca/μl. Concentration values are graphed logarithmically and time linearly.

Fig. 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 containing 5 × 106 cpm in 0·61 μmole 45Ca2+. After 48 h the labelled hydra were placed in normal non-radioactive medium. At various intervals during both the uptake and release phases, 2–3 hydra were removed and analysed, and cpm/o.D. values were calculated. Since 45Ca2+ uptake will be proportional to the difference between the [45Ca] in the medium and that in the hydra at any given time, Co is expressed as cpm 45Ca per O.D. unit, or l·46×cpm 45Ca/μl. Concentration values are graphed logarithmically and time linearly.

Fig. 5.

The steady-state ratio of Ca2+ in hydra to Ca2+ in the medium at various initial Ca2+ in the medium. Eight groups of about 15 cleaned H. littoralis were each suspended in 2 ml normal medium except that the initial CaCl2 concentration varied from 3 × 10–6 M to 0 ·05 M. All solutions contained 2·5 × 106cpm 45Ca per ml. At 18 h (O), 47 h (?) and 114 h (△), samples of 2–3 hydra were washed and analysed. On the basis of cpm/o.D. values, calcium concentrations were calculated. Hydra tolerated 0· 01 M-CaCI2, but not higher concentrations. 45Ca values below 10–5 M are somewhat unreliable because of ion leakage from hydra.

Fig. 5.

The steady-state ratio of Ca2+ in hydra to Ca2+ in the medium at various initial Ca2+ in the medium. Eight groups of about 15 cleaned H. littoralis were each suspended in 2 ml normal medium except that the initial CaCl2 concentration varied from 3 × 10–6 M to 0 ·05 M. All solutions contained 2·5 × 106cpm 45Ca per ml. At 18 h (O), 47 h (?) and 114 h (△), samples of 2–3 hydra were washed and analysed. On the basis of cpm/o.D. values, calcium concentrations were calculated. Hydra tolerated 0· 01 M-CaCI2, but not higher concentrations. 45Ca values below 10–5 M are somewhat unreliable because of ion leakage from hydra.

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

Since an appreciable portion of 45Ca was retained in the hydra for relatively long periods, groups of about 50 H. littoralis were labelled by being incubated in 2 ml of medium containing 4× 10–6 M-45CaCl2 with 5× 106cpm for 48 h, and grafts were made. The transfer of labelled calcium, expressed as R values in H1*/2F and HB/56F* grafts is given in Table 2. Clearly 45Ca moved much more rapidly than did thymidine. Transfer did not occur to any appreciable degree, however, by release from the labelled section into the medium followed by uptake into the unlabelled section. Labelled and unlabelled pieces which were in apposition, but not grafted together, for example, had R values of less than 0·1 even at 44 h. Furthermore, unlabelled hydra placed together with labelled grafts took up essentially no radioactivity (R < 0·3). Although 45Ca was lost progressively from all pieces during incubation, the specific activity of the initially labelled sections of grafts at the time of analysis varied from 4000 to 6000 cpm/o.D. unit for short incubations, and from 300 to 1500 cpm/o.D. unit for incubations of 19 h or more.

Table 2.

The transfer of 45Ca2 from labelled to unlabelled sections in grafts of H. littoralis

The transfer of 45Ca2 from labelled to unlabelled sections in grafts of H. littoralis
The transfer of 45Ca2 from labelled to unlabelled sections in grafts of H. littoralis

Transfer of 32P-labelled compounds from initially labelled to unlabelled sections in grafts of hydra. When H. littoralis was incubated in normal medium containing labelled inorganic phosphate, the 32P uptake into hydra was initially rapid and then plateaued between 1 and 2 days. Each major section of hydra equilibrated in this way had the same specific activity, ± 12 %. The 56F section was always somewhat more strongly labelled than sections H and 12 (0·01 < P < 0·05). Upon placing these 32P-labelled hydra in normal medium containing nonradioactive inorganic phosphate, approximately one-third of the radioactivity was released within 12–24 h, but thereafter very little radioactivity was lost during a subsequent 4-day period. For grafting experiments about 10 H. attenuata were injected with approximately 0 · 2 μ I. of 32PO4 solution which contained 2 μCi and 20 pg PO4 per μl. The hydra were incubated in normal medium containing 10–3 M phosphate overnight and then were grafted in the conventional way.

As shown in Table 3, significant radioactivity was transferred to the most distant slice within one hour of grafting. The R values steadily increased with time and by 46 h all sections had R values of 49 or higher. As in other cases, R values for grafts which did not hold together, or for pieces of unlabelled hydra which were incubated together with highly labelled hydra, were 0·4 or less during 5 h of incubation. In all experiments with 32PO4, the initially labelled piece had a specific activity of 5000–20 000 cpm/o.D. unit at the time of analysis. Similar experiments with H. littoralis, in which labelling was achieved by soaking in a solution containing radioactive phosphate for 1–2 days, gave essentially identical results.

Table 3.

The transfer of radioactivity from 32PO4-labelled to unlabeled sections in grafts oflA. attenuata

The transfer of radioactivity from 32PO4-labelled to unlabeled sections in grafts oflA. attenuata
The transfer of radioactivity from 32PO4-labelled to unlabeled sections in grafts oflA. attenuata

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

Transfer of 45Ca and32P phosphate in other types of grafts. Pieces of hydra labelled either with 45Ca or with 32PO4 were grafted in various combinations with unlabelled sections, and the grafts were incubated for 20 h. As shown in Table 4, the transfer of 32P-labelled compounds from labelled to unlabelled sections was roughly the same, regardless of the nature of the graft. Although showing greater variability, 45Ca was also transferred well from labelled to unlabelled pieces in all grafting combinations.

Table 4.

Transfer of radioactivity from 45Ca2+-or 32PO4-labelled to unlabelled sections in grafts of H. littoralis incubated for 20 h

Transfer of radioactivity from 45Ca2+-or 32PO4-labelled to unlabelled sections in grafts of H. littoralis incubated for 20 h
Transfer of radioactivity from 45Ca2+-or 32PO4-labelled to unlabelled sections in grafts of H. littoralis incubated for 20 h

In order to calculate approximate specific activities in hydra sections, it was useful to develop a rapid, sensitive and reproducible method for determining the cell mass of hydra. Sonoration of whole hydra, or parts of it, in a small volume for as little as 10 s yielded a relatively stable turbid solution, whose optical density at 250 nm was found to be proportional to the number of hydra present. Since one would expect hydra to be uniformly labelled when either 32PO4 or 45Ca is employed, the observed constancy of the specific activity, expressed as cpm per O.D. unit, in different parts of the hydra, suggests that the procedure does indeed give a practical measure of hydra mass. Other methods which have been used for expressing the cell mass of hydra are total nitrogen, DNA or protein values (Clarkson, 1969 a, b). Techniques for determining these compounds, however, are more time-consuming and involved than the simple turbidity-absorption procedure described here. Expressing data in terms of the number of hydra is of course quite misleading, since hydra vary greatly in size depending on species, nutrition and stage of maturation.

With respect to isotopic measurement, the direct counting by scintillation spectrophotometry of sonorated suspensions of hydra in a scintillation solution which tolerates appreciable quantities of water was rapid and adequately precise. Autoradiography, although useful in localizing isotopic compounds in cells or tissues of hydra (Campbell, 1965), suffers from the disadvantages that watersoluble materials are usually lost, the time lag between experiment and evaluation is long, and grain counting is tedious.

The fact that tritiated thymidine, presumably incorporated solely into the DNA of cells, does not move readily from labelled to unlabelled sections was more or less expected. Certainly no obvious movements of radioactivity occurred within 5 h, and a small but significant transfer of thymidine from the labelled to the initially unlabelled section occurred only after 23 h. This transfer of radioactivity is almost certainly due to the migration of cells. Although the mitotic index of cell division is more or less uniform throughout the hydra (Campbell, 1967 d) cells tend to migrate from the 12 region of the neck either down the gastric column or up towards the tentacles (Campbell, 1967 ó). About 8 days are required for individually marked cells to travel from the neck down into the budding region. The present finding that 1·4 to 5 % of the [3H]thymidine labelled compounds move from the H12 region into the gut section in one day is roughly in keeping with Campbell’s earlier studies with hydra marked with dye (1967 b). Also the fact that radioactivity moves less readily from the foot up into the 34B region accords with Campbell’s observation that gastric region cells migrate predominantly towards the foot.

Calcium was much more mobile. Within 5 h an appreciable movement of isotope from labelled to unlabelled sections occurred, and within 44 h all sections were significantly labelled. Clearly calcium transfer was much more rapid than cellular movement, and occurred at roughly the same rate from head to foot as in the opposite direction. The initial uptake of 45Ca () and its phase of rapid release (– = 6 h) generally agree with similar studies on other ions. 24Na, 42K and 82Ba, for example, are taken up by Pelmatohydra oligactis with half-lives () of roughly –3 h, whereas the t for the release of 24Na was approximately 5 h in distilled water (Lilly, 1955).

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

It is interesting to compare the rate of movement of 32P-labelled compounds with the time required for biological determination to take place in hydra. Signalling generally seems to be rapid, and in some cases determination occurs in about 1 h (Wolpert et al. 1971). For example, the time required for a 4 region to become determined as a foot is about 4 h in H1234 sections, but is only 1 h in 1234 sections. The simplest explanation for this observation is that some inhibitory signal from the head is transmitted to the 4 region within 1 h, thereby slowing the rate of foot determination (Wolpert et al. 1971). Although I do not suggest that phosphorylated compounds are necessarily involved in the control of pattern determination, it is interesting to note that only 32P-labelled compounds, of the three different types of isotopic compounds studied here, were transferred at a rate consistent with biological signalling times.

Polarity had little apparent effect on the transfer of either 45Caor 32P-labelled compounds from labelled to unlabelled sections of hydra. In reassembled hydra, like H12*/34B56F or H1234B/56F*, the rate of movement of radioactivity from head to foot and from foot to head is roughly the same. Similarly, in reverse-polarity grafts, such as H123*/321H and in reverse-polarity regenerating grafts such as 4321*/1234 the movement of both 45Caand 32P-labelled compounds was similar to that observed in reassembled hydra. These measurements are admittedly somewhat crude, and more precise analysis might yet show some polarity dependence. Furthermore, the movement of specific compounds of course might well be markedly influenced by polarity. It is interesting to note, however, that biological signals also seem to be transmitted readily in both directions with respect to a polarity gradient. For example, a certain symmetry of inhibition exists with respect to the head and foot regions, namely, that a head tends to inhibit the formation of a new head proximally whereas a foot tends to depress formation of a new foot distally (Wolpert et al. 1971). Furthermore, a hypostome transplanted into a digestive zone inhibits the formation of a new hypostome 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 grafts of hydra been studied (Hopper, 1962). Tentacles, H, or H123 sections were grafted laterally to the 56 region of host Pelmatohydra oligactis, where either host or graft was labelled, and the presence of 32P-labelled compounds was assessed qualitatively by autoradiography. Radioactivity was found to move from the labelled to the unlabelled portions within 6 h, was extensively distributed in 12 h, and was more or less equalized in 24 h. In all cases the movement 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 well with my own more quantitative observations.

Radioactive compounds might be transmitted in hydra by several possible ways: (1) by direct cell to cell transfer, (2) via the intercellular space (the mesoglia), (3) by circulation through the gut cavity, and (4) by interchange with the external medium. Of these four possibilities, only interchange with the external medium has been ruled out experimentally. Circulation through the gut cavity also seems unlikely, inasmuch as a gradient of radioactivity always existed from the initially labelled to the most distant unlabelled section. If compounds were extensively excreted into the gut cavity, the radioactivity in all initially unlabelled sections should be more nearly the same. Further studies on the mechanism of transfer would clearly be most welcome.

The chemical nature of the compounds which are transferred has not as yet been defined. Calcium, of course, might exist either as a free ion or as innumerable chelated forms. Tritiated thymidine is presumably incorporated largely into DNA, which probably accounts for its slow movement in grafts. When 32P is employed, over half of the radioactivity resides in the acid insoluble fraction of the initially labelled hydra. Since phosphate in most macromolecular and smaller components turns over rapidly, however, no deductions can be made about its transfer form.

In other studies various fractions derived from hydra have been shown to have inhibitory, and occasionally stimulatory, effects on regeneration (Webster, 1971). Except for the nematocyst toxin, tetramethylammonium, however, these compounds neither have been well characterized chemically, nor have been shown to possess specific pattern forming functions in hydra. It would be interesting, however, if some of these partially characterized morphogenic substances were 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 appreciation for the help of Miss Amata Hornbruch in maintaining and grafting hydra and to Professor Lewis Wolpert for providing facilities and critical encouragement.

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