J. CeU Sd. ai, I-13 (1976)

Printed in Great Britain

ELIMINATION OF HYDRA INTERSTITIAL AND

NERVE CELLS BY MEANS OF COLCHICINE

RICHARD D. CAMPBELLDepartment of Developmental and Cell Biology, University of California at Irvine,Irvine, California 92717, U.S.A.

SUMMARYHydra treated with colchicine or Colcemid become depleted of 95-99% of their interstitial

cells and derivatives of this stem cell: nematoblasts, nematocytes and nerve cells. A secondtreatment removes most or all remaining interstitial cells. The most effective treatment is an8-h immersion of whole Hydra attenuata in 004% Colcemid or 0-4% colchicine. Interstitialcells are eliminated through phagocytosis by both ectodermal and endodermal epithelial cells.The endodermal digestive cells send processes through the mesoglea which engulf interstitialcells and retract them into the endoderm. The resultant hydra, though devoid of nematocysts,can be artificially fed: these animals grow and bud and can be used to study the behaviourand development of tissue lacking nerve and interstitial cells.

INTRODUCTION

The freshwater hydra can grow perpetually (Brien, 1953) with new tissues andcells continually differentiating. Hydra also has remarkable regenerative abilities(Kanaev, 1969). This animal serves as a model for demonstrating the developmentalroles of different cell types.

Hydra's body wall consists of 2 epithelia (ectoderm and endoderm), each composedof a single type of self-renewing epithelial cell. However, the intercellular spaces ofthe ectoderm are crowded with undifferentiated interstitial cells. These cells are self-renewing and in addition differentiate into nerve cells, nematocytes (stinging cells)and nerve cells. We do not know whether the epithelial cells or the interstitial cells(and derivatives) are responsible for patterning the morphogenesis of hydra. Someevidence implicates interstitial cells in controlling pattern formation, because elimina-tion of the interstitial cells using either X-irradiation or the chemical nitrogen mustardseverely reduces the polyp's regenerative and budding abilities (Brien & Reniers-Decoen, 1955; Diehl & Burnett, 1964). However, hydra treated with these agentsinvariably die, suggesting that the epithelial cells are also damaged. Therefore theseagents are not specific enough to use in discovering cell functions.

In this report I describe a new method for eliminating interstitial cells from hydrawhich leaves the epithelial cells fully viable. Therefore it will allow one to studyhydra development in the absence of interstitial cells. This method involves treatinghydra with colchicine or Colcemid. I also describe the fate of the interstitial cellsduring treatment, as they are removed from the tissue through phagocytosis by theepithelial cells.

R. D. Campbell

METHODS AND MATERIALS

Hydra attenuata grown in M solution in mass culture (Lenhoff & Brown, 197c) were usedfor these experiments. Hydra were treated with various concentrations of Colcemid or colchicine,24 h after the most recent feeding, by pipetting animals into a solution of the chemical dissolvedin M solution. They were left in Petri dishes at densities of 5 hydra per millilitre, and at theend of the treatment they were rinsed copiously with fresh M solution. During the next2 days the hydra were rinsed twice per day; subsequently they were washed only once per day.

Treated hydra were force-fed Artemia nauplii by either pressing the nauplii through theirmouths using forceps, or by injecting nauplii through a polyethylene micropipette (with tipdiameter just equal to the width of a shrimp) inserted through the mouth.

Cell populations were analysed by phase microscopy of macerated tissue, as detailed byDavid (1973), and I use his terminology. Analyses were based on counts of at least 500 cellsper sample. Electron-microscopic observations were made on hydra fixed for 90 min in Msolution containing 0-4 % OsO4 and 1 % glutaraldehyde, and embedded in epoxy resin accord-ing to Spurr (1969).

Labelling with tritiated thymidine and subsequent radioautography were done according tothe methods of David & Campbell (1972).

RESULTS

Effects on hydra morphology

Photographs of hydra before, during and after an 8-h treatment with 0-4 % col-chicine are shown in Fig. 1 A-E. By the end of the treatment (Fig. 1 B) the tentacleshave become reduced and swollen; sometimes they are gone. The body is shortened,and many polyps lose their attachments to the dish. During the next several days allof these changes become accentuated until the hydra have lost most or all indicationsof a hypostome. The polyps swell up greatly. Many buds which were developingremain attached and do not subsequently detach except by gradual fission of themfrom the parent as a separate axis. In the first few days following treatment no newbuds are initiated; however on day 4 or 5 after treatment most hydra begin to budagain. The hydra are not fed in this interval, but well fed hydra normally will budfor a while after their last meal, and this process is apparently interrupted or delayedby the colchicine treatment. During the next few days tentacles are re-formed, theelongate form is restored, and the polyps eventually look rather normal (Fig. 1 E), buttheir tentacles are devoid of nematocysts.

A second colchicine treatment 10 days after the first results in a similar series ofchanges in hydra form (Fig. 1 F).

Time course of cell depletion

Fig. 2 shows a time course of the change in cell populations in hydra tissue followingan 8-h treatment with 0-06% Colcemid. The abundance of each cell type is represent-ed as the per cent contribution it makes to all the cells present. Interstitial cells andnematoblasts, which originally comprise 65% of all cells present, become reduced toless than 1 or 2% of the cells. Epithelial cells rise to over 90% of the cells, almostcertainly because the abundant interstitial cells are depleted. Nerve cells decline innumbers, but more slowly. Gland cells, comprising a few per cent of cells in untreatedand treated hydra, are not represented in Fig. 2.

Removal of Hydra interstitial cells 3

The interstitial cells and nematoblasts include several types of cells (David, 1973).The major remaining cell type after treatment is the 'big interstitial cell' (David,1973) which probably represents the undifferentiated stem cell (David & Challoner,1974; David & Gierer, 1974).

5 mm

Fig. 1 A-G. Effect of 8-h treatment with 0-4 % colchicine on Hydra attennata. Allphotographs made at same scale.

A, normal, untreated hydra.B, after 8 h in colchicine. Tentacles are bulbous and short.C, 1 day following treatment.D, 3 days after treatment.E, 2 weeks after treatment, without feeding.F, after 8 h in second colchicine solution.G, twice-treated hydra, fed for 2 months. Below is a recently detached bud.

This experiment has been repeated a number of times over the course of two yearswith consistent results. The left part of Fig. 4 illustrates the similar results of a sepa-rate experiment, using 0-4% colchicine in place of 0-06% Colcemid.

Dose dependency

Fig. 3 illustrates the effects of 8-h treatments in various concentrations of Colcemid.The levels of cell populations were determined 4 days after treatment. The levels ofthe different populations at this time are dependent on the strength of Colcemid

R. D. Campbell

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Time after Colcemid treatment, days

Fig. 2. Change in cellular composition following treatment of hydra with o-o6 %Colcemid for 8 h. Ordinate represents the per cent contribution of each cell type to thetotal cell population. Gland cells, not represented here, bring the summed percentagesto ioo. A, epithelial cells (D): B, interstitial cells and nematoblasts ( # ) ; C, nervecells (O).

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Fig. 3. Effect of an 8-h treatment with Colcemid at various concentrations on thecell population of hydra as measured 4 days later. The ordinate represents the percent which each cell type contributes to the total cell population. A, epithelial cells( • ) ; B, interstitial cells and nematoblasts ( • ) ; C, nerve cells (O).

Removal of Hydra interstitial cells 5

used; a maximal effect is shown at about 0-04% Colcemid. Concentrations of 0-08%and higher killed all the hydra treated: at 0-06% many hydra died.

Cell depletion shows a similar dependency on colchicine concentration except thatabout 10-fold higher doses are required; e.g. 0-4% colchicine gives an effect similarto 0-04% Colcemid.

Effect of a second treatment

A second colchicine treatment 2 weeks after the first results in the depletion of mostor all the remaining interstitial cells and nerve cells (Fig. 4). For example, 10 daysafter the second colchicine treatment no nerve cells or interstitial cells were seen in atotal of 2000 cells counted.

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Fig. 4. Cellular composition of hydra subjected to 2 successive 8-h treatments(arrows) with 0-4 % colchicine. The ordinate is a logarithmic plot of the ratio of nervecells to epithelial numbers ( # • ) or the ratio of interstitial cells, nematoblastsand nematocytes, to epithelial cell numbers (O O)- On days 24 and 25, a total of2000 cells were counted in 3 hydra.

6 R. D. Campbell

Effect of feeding treated hydra

Hydra treated once or twice with colchicine will grow and bud rapidly if force-fed.Their tentacles become long and slender. A few hydra treated once with colchicinehave some nematocysts in the tentacles; most have none. I have never observed asingle nematocyst in a tentacle of the many hydra I have treated twice with colchicine(or Colcemid); even after growth and budding for months, these hydra lack nemato-cysts.

Hydra that have been treated twice with colchicine (hereafter called 'depleted'hydra) can be force-fed. Although many die initially, those which survive grow to anormal size and maintain that size while budding. Fig. i G shows a depleted hydra.In general their morphology is strikingly normal. But some particular features areabnormal. The stalk tends to grow longer continuously, and in several cases a newbase is formed in the central stalk and the stalk fragmented. The gastric region re-mains swollen. The hypostome is not as well defined, and the tentacles become morenumerous and irregularly spaced than in normal hydra (see the detached bud inFig. IG). Buds remain adhering to the parent stalk for days or weeks, although theirbasal disks form normally.

The behaviour of treated hydra is abnormal. They seldom attach to the dish; thebasal disk is well developed but is covered by an exudate. The hydra do not exhibita feeding response to glutathione (W. Heagy, personal communication). They havelittle spontaneous contractile activity, and never open their mouths widely. Probablyfor this reason, they are perpetually bloated, because hydra tissues are thought toaccumulate water osmotically in their gastric cavities (Macklin & Josephson, 1971).They do collapse occasionally by extrusion of fluid from the mouth, but indigestibleparticles (e.g. Artemia egg cases) remain in the gastric cavity for days. Furtherobservations of the behaviour and electrophysiology of depleted hydra will be re-ported elsewhere (Campbell, Josephson, Schwab & Rushforth, in preparation).

Survival of depleted hydra

Depleted hydra which were not fed survived for weeks, getting continually smaller(although unusually slowly), as do normal, starved hydra. But when they were fed,survival was not as good as in normal animals fed regularly. Two problems wereencountered in keeping fed, twice-treated hydra alive:

(a) Only a fraction (about 1 out of 20) of the hydra which I attempted to feedsurvived for more than a week. This was probably due to the great difficulties inforcing food into such small polyps; most were severely torn repeatedly. This initialloss of most hydra means that survivors were a highly selected set of individuals,which may be of importance in explaining their later growth (see below).

(b) Those which did survive initial feeding grew well; however, these tended todie during the next several weeks. Tentacles shortened and became irregular, muchdebris accumulated in the gastric cavity, and opaque masses of bacteria accumulatedwithin and between cells. Finally the hydra rapidly decreased-in size and disintegrated.

I interpret this lack of survival as due to bacterial infection, which was visible; it

Removal of Hydra interstitial cells 7

may be that some of the behaviour patterns (such as movements or more frequentvoiding of the gastric cavity) which were eliminated by colchicine normally havecleansing functions.

When the hydra that survived initial feeding were grown in the continued presenceof antibiotic (50 /*g/ml Rifampicin) they survived well, some budding for months ofculture.

Specificity of colchicine treatment

The colchicine (or Colcemid) treatment eliminates most non-epithelial cells.Apparently the epithelial cells are not permanently damaged because the hydra growand bud. In order to analyse the cycling rates of the epithelial cells, I began feedinghydra 10 days after a single treatment and began continuously labelling them with[3H]thymidine 4 days later. Hydra sampled at 4 daily intervals during the continuouslabelling showed that the epithelial cells were cycling with kinetics similar to those innormal hydra (described by David & Campbell, 1972); Fig. 5 shows that after 3 daysof labelling, three-quarters of the epithelial cells had become labelled. Thus most or allepithelial cells continue to proliferate.

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continuous labelling, days

Fig. 5. Per cent of epithelial cells labelled by continuous application of ['H]thymidine.These hydra had been treated for 8 h in 0-4% colchicine 14 days previously, andhad been fed daily for 4 days before labelling.

Histological aspects of cell depletion

During and following colchicine treatment, the interstitial cells are removed fromthe tissue through phagocytosis by epithelial cells. Most of the interstitial cells areengulfed by the endodermal epithelial cells which send processes through the mesogleaand draw the interstitial cells into the endoderm.

The histological sections shown in Fig. 6A-F are from the gastric regions of hydra,sectioned transversely to the body axis. Control hydra (Fig. 6A) have the 2 tissues,

R. D. Campbell

Removal of Hydra interstitial cells 9

ectoderm and endoderm, separated by a thin (1- to 2-/im), acellular mesoglea. Themesogleal surface is entirely lined by the epithelial cells of the 2 layers. The endoder-mal epithelial cells (also called digestive cells) have broad basal surfaces which coverthe mesoglea (see Fig. 6 A).

The ectodermal epithelial cells completely line the outer surface of the mesoglea.This lining is composed of a continuous sheath of parallel, axially oriented contractileprocesses extending from the cells. There is no large, flat base to the cell as there isto digestive cells.

In the gastric region of control hydra there are a few fine epithelial cell processeswhich traverse the mesoglea (they are more abundant in other parts of the hydra);these processes are usually less than 1 fira in width, and often meet midway throughthe mesoglea with an opposing process from the other tissue layer.

Interstitial cells, nematoblasts, migrating nematocytes and nerve cells are almostexclusively located in the ectoderm, above the mat of muscle processes. They hardlyever contact the mesoglea. They are wedged between adjacent epithelial cells, andwithin the crypt-like spaces defined by the stalks descending from the epithelial cellbodies down to the sheath of contractile processes.

Within 2h after the beginning of colchicine (Fig. 6 B) or Colcemid (Fig. 6 c)treatment, large numbers of interstitial cells and nematoblasts are in the endoderm.Some cells can be seen traversing the mesolamella (arrows, in Fig. 6B, C). These cellsare inside digestive cell processes which penetrate into the ectoderm (Fig. 7). Engulf-ment by digestive cells has the following characteristics. The cytoplasm of the diges-tive cell process is finely granular and homogeneous, containing no organelles. Theportion surrounding the interstitial cell is thin and uniform. The digestive cellprocess penetrates between adjacent ectodermal muscular processes. The entireprocess containing the ectodermal cell is withdrawn into the endoderm.

Fig. 6. Effect of colchicine treatment on the histological appearance of hydra. Allphotographs are at same magnification (see c for scale), and are i-/tm Epon sectionstransverse to the body column. In all micrographs, the ectoderm is at top, the endo-derm and gastric cavity at bottom, with the mesoglea between.

A, normal hydra. Numerous interstitial cells are wedged between the vacuolatedectodermal epithelial cells.

B, after 2 h in 0 4 % colchicine. Many interstitial cells and nematoblasts are nowpresent in the endoderm. Arrow points to a small cell (perhaps a nerve cell) crossingthe mesoglea.

C, after 2 h in 0-04 % Colcemid. Many interstitial cells and nematoblasts are in theendoderm. Arrow points to a nematoblast crossing the mesoglea. Some of thecellular debris free in the gastric cavity is visible at the lower right.

D, after 8 h in 0 4 % colchicine. Almost all interstitial cells and nematoblasts arein the endoderm and in the cellular debris floating in the gastric cavity (just visibleat bottom of micrograph).

E, 15 days after 8-h treatment in 0 4 % colchicine. At the left is an ectodermalepithelial cell nucleus.

F, starved, untreated control hydra. Ectodermal epithelial cell nucleus is visible atright; towards the left are several interstitial cells.

10 R. D. Campbell

Fig. y. Hydra after 2 h in 0 4 % colchicine. An ectodermal nematoblast (top) issurrounded by a process of an endodermal epithelial cell ('digestive cell'). This samedigestive cell also contains remnants of 2 other non-epithelial cells. Adjacentepithelial cells (to the left and below) contain a variety of nematoblasts in variousstages of degeneration.

Removal of Hydra interstitial cells 11

In entire cross-sections of hydra, about two such processes exist per section, yetscores of interstitial cells, per section, are transported within a few hours. Thereforethe processes must form, engulf and retract rapidly, within a span of a few minutes.I did not see any cells traversing the mesoglea without being inside an endodermalcell process.

Engulfed cells usually, but not always, look abnormal. Generally the cytoplasm isvacuolated.

Other tissue abnormalities arise during colchicine and Colcemid treatment. In theectoderm, muscular processes sometimes separate from one another, the processesthemselves retract or somehow diminish in size (this is most easily seen in maceratedtissue) and large intercellular spaces sometimes appear. These changes occur pro-gressively during the treatment. The endodermal cells become filled with interstitialcells and nematoblasts in various states of digestion (Figs. 6B-D, 7), and large amountsof debris fill the gastric cavity (Fig. 6 c, D). This debris appears to represent portionsof digestive cells, crowded with interstitial cells, which have broken free of the tissue.

Microtubules are present throughout the 8-h treatment. They are present in the3 classic locations for hydra microtubules: within nerve cell processes; supporting thecnidocil apparatus in nematocytes; and ensheathing the developing nematoblastcapsule.

Ten days after treatment, at which time the hydra look more or less normal, theectoderm is nearly lacking all cells except the epithelial cells (Fig. 6E). The epithelialcells differ slightly in appearance from those of normal hydras: the nuclei are irregularin shape, and the cytoplasm is more vacuolate. Fed and growing hydra which havebeen treated twice have a similar appearance. Starved, untreated control hydra(Fig. 6F) have some of these abnormal appearances.

DISCUSSION

Colchicine and Colcemid can be used to deplete Hydra attenuata of interstitialcells, nematoblasts, nematocytes and nerve cells. The optimal initial treatment is for8 h in 0-04% Colcemid or 0-4% colchicine followed by a week of culture.

Depletion is mediated mainly by digestive cells which send phagocytic processesthrough the mesoglea into the ectoderm. Conceivably this is a normal process, andcolchicine renders the interstitial cell types susceptible to phagocytosis. We often seeevidence of cellular remains inside unbroken epithelial cells separated by macerationof normal hydra. Thus, hydra epithelial cells may normally scour the intercellularspaces to remove damaged or unwanted material. Ectodermal epithelial cells rapidlytake up carbon particles and other materials injected into the intercellular spaces(Campbell, 1973). Therefore, hydra epithelial cells are strongly phagocytic at theirlateral surfaces.

Conceivably, other reports of cells traversing the mesoglea in hydra (see Schlottke,1930; Noda, 1968) might reflect engulfment and transport by digestive cells. Thismay have been missed previously because the digestive cell process is so thin that itis difficult to see by light microscopy (see Fig. 6c).

12 R. D. Campbell

Although the effects of colchicine (or Colcemid) are usually ascribed to their effectson microtubules, in hydra they may be acting in some other way. Nearly all interstitialcells and nematoblasts, and many nerve cells, are removed during an 8-h treatment.But only about a third of the interstitial cells would normally have divided in thistime (Campbell & David, 1974) and the nematoblasts and nerves do not divide.Therefore colchicine is not acting specifically on the mitotic apparatus of cells. Also,microtubules are present during the entire treatment. Even microtubules in nerveprocesses, generally the most labile microtubules, were present. I did not make aquantitative study of microtubule abundance, however. The persistence of micro-tubules in these hydra is all the more surprising considering the high doses of col-chicine and Colcemid used. Burns (1973) reported that the binding of colchicine totubulin in a number of more-primitive organisms is low, suggesting that the col-chicine-binding site evolved significantly during metazoan evolution. A feeblecolchicine sensitivity is also suggested by the paucity of published reports usingcolchicine to study mitotic patterns in hydra (Sturtevant, Sturtevant & Turner, 1951;Corff & Burnett, 1969). I have had no success at accumulating dividing mitotic hydracells using colchicine.

A number of investigators have reported that colchicine has marked effects onhydra. At concentrations lower than those used here (as well as at these high concen-trations), colchicine and Colcemid prevent developing buds from separating from thecolony (Tammariello, 1969; Corff & Burnett, 1969). It can evoke additional hydranthstructures (Webster, 1967) or basal disks and peduncles (see Corff & Burnett, 1969).The latter authors also described the accumulation of interstitial cells and nemato-blasts in the endoderm, using lower concentrations of colchicine than used in thisstudy. Colchicine and Colcemid also affected graft stability and the formation ofsupernumerary structures (Shostak & Adams, 1975).

Since the treated hydra when force-fed can grow and multiply, they should serve asexcellent models for studying the developmental capacities of tissue composed only ofepithelial cells. However, whether or not hydra completely lacking interstitial cellsand nerve cells can be grown is yet to be established. Although the assayed twice-treated hydra had none of these cell types detectable (Fig. 4), those twice-treatedhydra which were successfully grown and later analysed still contained a few interstitialcells (about 0-3-1 per 100 epithelial cells) (Campbell, Josephson, Schwab & Rushforth,in preparation). The growth conditions may select for hydra with a few remaininginterstitial cells. Nevertheless, even those polyps are so abnormal in cellular compo-sition that they should prove useful in studying the roles of interstitial and nerve cells.

REFERENCES

BRIEN, P. (1953). La pe'rennite' somatdque. Biol. Rev. 28, 308-326.BRIEN, P. & RENIERS-DECOEN, M. (1955). La signification des cellules interstitielles des hydres

d'eau douce et le probleme de la reserve embryonnaire. Bull. biol. Fr. Belg. 89, 258-325.BURNS, R. G. (1973). aH-colchicine binding. Failure to detect any binding to soluble proteins

from various lower organisms. Expl Cell Res. 81, 285-292.CAMPBELL, R. D. (1973). Vital marking of single cells in developing tissues. India ink injection

to trace tissue movements in hydra. J. Cell Sri. 13, 651-661.

Removal of Hydra interstitial cells 13

CAMPBELL, R. D. & DAVID, C. N. (1974). Cell cycle kinetics and development of Hydraattenuata. II. Interstitial cells. J. Cell Sci. 16, 349-358.

CORFF, S. C. & BURNETT, A. L. (1969). Morphogenesis in Hydra. I. Peduncle and basal discformation at the distal end of regenerating Hydra after exposure to colchicine. J. Embryol.exp. Morph. 21, 417-443.

DAVID, C. N. (1973). A quantitative method for maceration of Hydra tissue. Arch. EntwMech.Org. 171, 259-268.

DAVID, C. N. & CAMPBELL, R. D. (1972). Cell cycle kinetics and development of Hydraattenuata. I. Epithelial cells. J. Cell Sci. 11, 557-568.

DAVID, C. N. & CHALLONER, D. (1974). Distribution of interstitial cells and differentiatingnematocytes in nests in Hydra attenuata. Am. Zool. 14, 537-542.

DAVID, C. N. & GIERER, A. (1974). Cell cycle kinetics and development of Hydra attenuata.III. Nerve and nematocyte differentiation. J. Cell Sci. 16, 359-374.

DIEHL, F. A. & BURNETT, A. L. (1964). The role of interstitial cells in the maintenance ofHydra. I. Specific destruction of interstitial cells in normal, asexual, and non-buddinganimals. J. exp. Zool. 155, 253-259.

KANAEV, I. I. (1969). HYDRA. Essays on the Biology of Fresh Water Polyps. [Translated byE. T. Burrows & H. M. Lenhoff; ed. by H. M. Lenhoff. Published by the editor], 452 pp.

LENHOFT, H. M. & BROWN, R. D. (1970). Mass culture of hydra: an improved method and itsapplication to other aquatic invertebrates. Lab. Animals 4, 139—154.

MACKLIN, M. & JOSEPHSON, R. K. (1971). The ionic requirements of transepithelial potentialsin Hydra. Biol. Bull. mar. biol. Lab., Woods Hole 141, 299-318.

NODA, K. (1968). Migratory cells traversing the mesoglea in Hydra. J. Fac. Sci. Hokkaido Univ.(ser. VI, Zoology), 16, 353-358.

SCHLOTTKE, E. (1930). Zellstudien an Hydra. I. Altern und Abbau von Zellen und Kernen,Z. mikrosk-anat. Forsch. 22, 493-532.

SHOSTAK, S. & ADAMS, J. A. (1975). Morphogenetic gradients in multiple-graft Hydra viridis.I. The effects of Colcemid and colchicine. J. exp. Zool. 192, 43-56.

SPURR, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron micro-scopy. J. Ultrastruct. Res. 26, 31-43.

STURTEVANT, F. M., STURTEVANT, R. P. & TURNER, C. L. (1951). Effect of colchicine onregeneration in Pelmatohydra oligactis. Science, N.Y. 114, 241-242.

TAMMARIELLO, R. V. (1969). The Action of Colcemid in Causing Bud Retention in Hydra viridis.Ph.D. Thesis, University of Pittsburgh, 69 pp.

WEBSTER, G. (1967). Studies on pattern regulation in Hydra. IV. The effect of colcemide andpuromycin on polarity and regulation. J. Embryol. exp. Morph. 18, 181-197.

(Received 26 September 1975)