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J. Cell Set. 29t 17-33(1978) 17Printed in Great Britain © Company of Biologists Limited 1978
DEVELOPMENT OF HYDRA LACKING NERVE
AND INTERSTITIAL CELLS
BEVERLY A. MARCUM* AND RICHARD D. CAMPBELLDepartment of Developmental and Cell Biology and Center for PatJiobiology,University of California, Irvine, California 92717, U.S.A.
SUMMARY
Hydra attenuata were rendered free of interstitial cells (I cells) and interstitial cell deriva-tives by colchicine treatment. These hydra were then cloned and cultivated for 18 months andtheir developmental capacities were studied. Some experimental hydra possessed a few (about1 % of the normal numbers) interstitial cells and retained this low level during prolongedculture and active growth without the differentiation of I-cells into specialized cells. Otherhydra were completely freed of interstitial cells by the colchicine treatment. Maceration andhistological analyses showed that once a hydra is freed of all interstitial cells it does not recoverthem, nor do its buds contain interstitial cells. I cell-free hydra also lack nerve cells, nemato-cytes, gametes and endodermal gland cells, and the tissue consists solely of ectodermal andendodermal epithelial cells. Hydra completely lacking interstitial cells grow, bud, exhibittissue renewal patterns, regenerate and preserve polarity generally typical of normal hydra.I cell-free hypostomal tissue has inductive capacity, as does normal hypostomal tissue, whenimplanted in I cell-free or normal gastric tissue. Regenerating I cell-free tissue undergoesprecocious determination as does normal tissue. Only in some quantitative aspects do I cell-free hydra develop abnormally. We conclude that hydra consisting only of epithelial cells arecapable of essentially normal development.
INTRODUCTION
Hydra tissues contain just a few cell types. Diverse current investigations are at-tempting to define the developmental and physiological roles of the different cells(Brien & Renier-Decoen, 1955; Lesh, 1970; Gierer et al. 1972; Schaller, 19760,6).There are 3 major self-perpetuating cell types in hydra: ectodermal epithelial cells,the endodermal epithelial cells and the interstitial cells (David & Campbell, 1972;David, 1973; Campbell & David, 1974). We are eliminating from hydra one of themajor classes of cells, the interstitial cell and its derivatives, and testing the develop-mental capacities of the remaining tissues which are composed solely of epithelial cells.
Interstitial cells are found mainly in the ectoderm, and are the stem cells for all ofthe nerve cells, nematocytes and gametes (David & Gierer, 1974). These differentiatedand specialized cells do not themselves divide. Therefore, by eliminating the inter-stitial cells, one can effectively and permanently prevent the formation of nerve cellsand other interstitial cell products.
In this paper we describe a method for eliminating interstitial cells and their deriva-tives, making it possible to study the development and physiology of hydra which are
• Address for correspondence: Dr Beverly Marcum, Center for Pathobiology, Universityof California, Irvine, Irvine, California 92717, U.S.A.
18 B. A. Marcum and R. D. Campbell
free of both nerve and interstitial cells, 2 cell types long implicated as crucial to hydradevelopment (Tardent, 1954; Lentz, 1966; Schaller, 1973). We also report here thathydra composed only of epithelial cells have extensive developmental capacities.
MATERIALS AND METHODS
Experiments described here were carried out with a strain of normal Hydra attenuata (Pall.),obtained from Professor A. Gierer, which was originally collected by Professor P. Tardent inLake Zurich in 1962. This is the same hydra population that has been used extensively in otherresearches (for example, Bode et al. 1973; Tardent, 1966; Tardent & Morgenthaler, 1966).The methods described here do not have the same effects on several other strains and speciesof hydra.
Colchicine (obtained from Sigma Corp., and kept under refrigeration as powder) treatmentconsisted of pipetting hydra into Petri dishes containing 0 4 % colchicine dissolved in M solution(containing NaCl instead of NaHCO,, Muscatine & Lenhoff, 1965). After 8 h the hydra werewashed repeatedly and washing was repeated several times daily thereafter for 1 week. Thesame animals were retreated with colchicine after 10-20 days. The entire regime described herewill be termed a 'double colchicine treatment.' All subsequent culturing was done in the sameM solution containing 50 fig/ml rifampicin. Except for a 3-day recovery period following eachtreatment, the hydra were fed daily. Feeding was accomplished by inserting freshly killedArtemia nauplii through the hydra's mouth, using a finely drawn polyethylene mouth pipetteto manipulate the shrimp. As many shrimp as possible, usually 1-8, would be fed to a hydra.New buds must be fed before detachment or else they become too small to feed. Between 8 and12 h after a meal each gastric cavity is well flushed with a jet of culture solution by means of apolyethylene micropipette inserted through the hydra's mouth. The hydra do not survivewithout this treatment.
Maceration and cell identification followed the procedures of David (1973). Interstitial cellrepopulation was accomplished by grafting a top or bottom half of a vitally marked, normalhydra to the complementary piece of a colchicine-treated polyp. Several days later the normaltissue was cut away using the vital marking as a guide. Vital marking was carried out by themethods of Campbell (1973), using small spots of injected ink for studying tissue movementsand large spots, extending around the circumference of the hydra, in experiments involvinggrafting. Marking was done 3 days prior to grafting.
RESULTS
Elimination of interstitial cell classes
A double colchicine treatment results in near elimination of all cells except theepithelial cells. Fig. 1 illustrates the time course of relative cell abundance changes inresponse to colchicine treatment. Interstitial cells, nematoblasts and nematocytesbecome depleted within a few days after each colchicine treatment. Nerve and glandcells decline in numbers somewhat more slowly. These kinetics are highly repro-ducible with this strain of hydra (compare with fig. 4 in Campbell, 1976).
After 2 weeks following the second colchicine treatment, few non-epithelial cells arepresent and the cell population levels do not show extensive further changes. Moreextensive cell counts (Table 1) indicate that hydra sampled at this time contain alow average number of non-epithelial cells and that this number varies somewhatfrom one preparation to the next.
From a population of twice-treated hydra we cloned 13 individuals (methods forfeeding and culturing these hydra are described under Materials and methods). Wheneach clone consisted of several hydra we began to analyse the cell compositions of
/ cell-free hydra
60
8 ^12 16 20 24
Days after first colchicine treatment
r28
Fig. 1. Time course of cell disappearance following colchicine treatment. Thepopulation levels of nerve cells ( •—•) , interstitial cells (O O) and endodermalgland cells ( x x ) are expressed relative to epithelial cell number. Colchicinetreatments (0-4 %, 8 h) were given on days o and 11 (arrows).
Table 1. Number of nerve and interstitial cells present 2 weeks afterthe second colchicine treatment
•Experimentno.
No. ofhydra
No. of cellssampled
No. ofnerve cells
No. ofinterstitial
cells
123
Total
312
12
1500
12000
6000
oo55
o9
27 19500 5 17
Each experiment involved independent colchicine treatment.
individual polyps. Table 2 shows cell counts in individual polyps of these 13 clones.At the level of precision of these counts, the 6 clones listed at the top of Table 2lacked interstitial cells and interstitial cell derivatives.
Three of these clones were then propagated for over a year, and more extensive cellcounts (Table 3) were made at intervals during this period.
Apparently 2 of these clones (IE and IIA) are completely free of all cell typesexcept epithelial and gland cells. In one individual of clone IIK one pair of large(David, 1973) interstitial cells were observed in 3000 counted cells but no interstitialcell derivatives (nerve cells or cells of the nematocyte lineage) have been seen.
20 B. A. Marcum and R. D. Campbell
A small number of structures in our maceration preparations are not interpretable;these are recorded in the right-hand column of Tables 2-4. None of these structureslooked like nerve or interstitial cells, and we have not been able to prepare macerationswithout them. These bodies may represent degenerating cell fragments arising from
Table 2. Relative cell compositions of polyps in 13 clones of colchicine-treated hydra
Clonelabel
IE
IIA
II K
II LHIDIIIEI K
ILI I C
H E
IIIA
IIIB
IIIL
Epithelial
966957
1171
9 5 i952
95O952
961
952
959
875656
943
948
973949
1352
95495O962
9 0 0
Biginterstitial
0
0
0
0
0
0
0
0
0
0
2
0
2
1
342
0
1
2
2
Cell
Nerve
0
0
0
0
0
0
0
0
0
0
00
0
0
0
0
0
0
0
0
0
counts
Nematocytelineage
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
00
0
Gland
344 0
62
4348
5646
3948
39
5541
55
51
4444
7445463698
Unrecog-nizable
0
3
1
60
0
2
1
1
2
430
0
0
32
1
30
0
Each line represents a separate polyp. Cell types are labelled according to David (1973)except: 'Nematocyte lineage' includes all little interstitial cells, nematoblasts and nemato-cytes; ' gland' includes all endodermal glandular cells and ectodermal basal disk epithelialcells; 'unrecognizable' includes structures resembling cells but not fitting into the othercategories.
endodermal cell sloughing at the basal disk and tentacle tips, and are visible in histo-logical sections. However, since it is conceivable that they are aberrant nerve or inter-stitial cells, and unrecognizable, this debris puts a limit on cell determination by themaceration method. We conclude that within the limits of the maceration method,there are no non-epithelial cells in clones IE and IIA.
Therefore we conclude that a double colchicine treatment removes most but notall interstitial cells and interstitial cell derivatives. Those which do remain, however,are not equally distributed among hydra. Some hydra entirely lack interstitial, nema-toblast, nematocytes and nerve cells, and these hydra can be selected and cloned as
/ cell-free hydra 2 1
Table 3. Relative cell compositions of 3 clones of hydra treated twice
with colchicine
Clone
I E
IIA
U K
ControlJ
Epi-thelial
962956971
936925
9741909979
19899 9 0
2712
11519519499 0 0
95O966
19354568
9 9 2
2993
95°956958952952979
2934978
2686
2 5 93 1 8
(29%)
Biginter-stitial
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
2 4 2186
(21 %)
Cell counts
Nerve
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4337
(4%)
Nema-tocyte
lineage
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
39O4 0 1
(4O%)
Gland
34432 9587 21 0
81
2 40
0
0
62
4351
9347326 2
0
0
0
5°434 2
48482 1
640
0
6658
(6-2 %)
Unrecog-nizable
41
0
6
3161 0
0
1 1
1 0
9
1
60
732
34 2
87
0
1
0
0
0
0
0
2 2
14
Date#
day, month, year
5/i i/7526/11/75
2/1/763/i/763/1/76
23/1/7618/3/7621/4/76i9/8/76t23/8/7614/4/77
29/10/756/11/75
26/11/7520/12/7523/12/7513/3/7620/4/76i9/8/76t23/8/76
3/3/77
6/1i/7526/11/7530/12/75
1/1/761/1/76
18/3/7621/4/7623/8/76
3/3/77
See Table 2 for explanation of column headings. After 21/4/76 ' Gland' refers only to endo-dermal gland cells.
• Colchicine treatment was begun on 23/9/75.t IE and I IA totals for 19/8/76 were obtained from 2 and 4 polyps, respectively. All other
totals are from single polyps.% Extensive normal cell counts are presented in Bode et al. (1973).
22 B. A. Mar cum and R. D. Campbell
polyps consisting only of epithelial cells. Once hydra are free of interstitial cells, theyremain free of interstitial cells through extensive growth, budding and culturing overa period of many months.
Histological evidence that some clones lack interstitial cells
To confirm our maceration counts we prepared cloned colchicine-treated hydra forhistological examination. Two animals from each of 3 clones (IE, IIA and IIK)were serially sectioned transversely and each cell identified and counted. No inter-stitial cells, nerve cells, or cells of the nematocyte lineage were found. The detailsof the histological analysis will be described elsewhere.
Interstitial cells in low numbers
We have some clones of hydra which have low numbers of interstitial cells. Thesehave been produced by either single colchicine treatment or by partial repopulation ofcolchicine-treated hydra through temporary implant of normal tissue. Table 4 illus-
Table 4. Relative cell composition of hydra in clone no. 46-1 *, containing relativelyfew interstitial cells
Epithelial
1216969979993985
2731
1982
Biginter-stitial
17
1713
58
1318
Cell counts
Nerve
0
0
0
0
0
0
0
Nema-tocytelineage
0
0
00
0
0
0
Gland
8 0
2300
01
0
Unrecog-nizable
2
1 0
82
60
0
Date,day, month, year
4/"/7S10/5/76
29/7/762/8/763/8/76
25/10/76
5/2/77
See Table 2 for explanation of column headings. After 10/5/76 'Gland' refers only to endo-dermal gland cells.
• The original polyp of this clone was a twice-treated individual repopulated with a smallnumber of interstitial cells from a temporary graft of normal hydra tissue on 3/10/75.
trates 3 unusual characters of such polyps, as exemplified by our clone number46-1. First, interstitial cells do not increase in number and thus repopulate the hydra.(At interstitial cell levels of greater than several percent of normal, repopulationoccurs.) Second, no nerve, gland, or nematocyte cells are found in these hydra. Whenonly a small number of interstitial cells remain in an animal, these cells do not differen-tiate. Third, hydra containing low levels of interstitial cells have the morphologicalappearance of hydra which are completely free of interstitial cells.
Thus polyps retaining a few interstitial cells can be propagated indefinitely in acondition free of specialked cells.
/ cell-free hydra 23
Viability of I cell-free hydra
In this paper we use the term 'I cell-free hydra' to indicate hydra treated twicewith colchicine and subsequently propagated asexually, and which are apparentlycompletely free of interstitial, nematoblast, nematocyte, endodermal gland andnerve cells. These hydra consist of only epithelial cells. Therefore, the term ' I cell-free' is a convenient label for a condition broader than simply lacking interstitialcells.
I cell-free hydra grow if fed. When buds detach and reach full size they cannot bedistinguished from the parents. Three clones (I E, IIA and III E) have been extensivelycultured for over a year and have produced more than 500 polyps. However, thisnumber does not represent the maximum possible number of hydra obtainable sincemany polyps were used for experiments, Thus, these hydra are capable of extensiveand probably unlimited growth, as are normal hydra (Brien & Reniers-Decoen, 1949).
I cell-free hydra were repopulated with normal interstitial cells by means of atemporary tissue implant (see Materials and methods). Repopulated hydra appearnormal in morphology, growth, budding and behaviour. Since no new epithelial cellsare introduced by this repopulation procedure, we conclude that the epithelial cellof colchicine-treated hydra are viable and unimpaired by the colchicine treatment.
Slow changes in I cell-free hydra
I cell-free hydra change in appearance over the course of the first several monthsof culturing. Initially they are less swollen and more coloured than later. Initially theyhave greater digestive capacities, as evidence by extensive breakdown of the shrimpwhich are pressed into their gastric columns. After a few months, shrimp are unchangedin appearance after remaining in the gastric cavity for a day or longer. After prolongedculture, I cell-free hydra tissue becomes less resistant to stretching and less coloured byfood. These long-term changes in animals take place after interstitial cell elimination.
Morphology of I cell-free hydra
I cell-free hydra have all the morphological regions of a normal hydra, but the bodyproportions are atypical.
Fig. 2 shows normal and I cell-free hydra. I cell-free hydra have swollen distalcolumns, narrow peduncles and small basal disks. The basal disks are sticky and accu-mulate white envelopes, presumably through secretory activity, but the animals donot attach to the substrate. Buds remain perched on parent stalks for many days. Thelower part of the body is frequently annulated. The tissue is nearly colourless, trans-parent and stretched. The hypostome is large, flat and thin rather than being athick conical protrusion as in normal hydra. I cell-free hydra have tentacles that aremore numerous than normal and are often of different lengths. Tentacles may beirregularly spaced but remain more or less in a whorl. The tentacles are also shorterthan normal and are straight and slender. The morphology of I cell-free hydravaries with feeding. In one set of hydra fed regularly the tentacle number averaged9-6, but after 2 weeks of starvation the average number of tentacles increased to
34 B. A. Marcum and R. D. Campbell
13 per animal. Multiple hypostomes appear frequently in starved I cell-free hydra(Fig. 2 c). Under poor culture conditions (for example, if the gastric cavity is notflushed following feeding) the hydra may quickly shrink and die; the first sign ofsuboptimal conditions is a shortening and kinking of the tentacles (Fig. 2D).
Fig. 2. Appearances of a normal hydra and of colchicine-treated hydra grown for overa year without interstitial cells or derivatives, A, normal Hydra attenuata; B, I cell-freehydra; c, I cell-free hydra starved for 10 days, showing extra hypostome; D, I cell-freehydra showing first signs of response to inadequate growth conditions.
Behaviour of I cell-free hydra
I cell-free hydra lie motionless on their sides. They show no spontaneous activitythat can be observed directly. They do not show any response to reduced glutathioneadded to the culture solution (W. Heagy, personal communication).
Some spontaneous activity can be observed in time-lapse movies of I cell-freehydra. Once or several times a day the fluid in the gastric cavity discharges suddenlyinto the culture and the hydra collapses. Re-extension follows. Slight columnpulsing and tentacle bending at their junction with the body occurs at irregularintervals. Annulations on the column propagate upwards along the column. All ofthese activities are more pronounced following a meal.
Strong stimulation, such as poking, will cause contraction of the polyp and tentacles,followed by gradual re-extension. The basal disk is the most sensitive part of thehydra to such stimulation, but it is much less sensitive than that of a normal hydra.Further behavioural and electrophysiological characteristics of colchicine-treatedhydra are reported elsewhere (Campbell et al. 1976).
Development of I cell-free hydra
Budding. I cell-free hydra bud at rates which are within the normal range for hydra.Budding rate is affected markedly by feeding rate. Table 5 shows the budding rates
for I cell-free hydra fed 8 shrimps each day and the budding rates of normal controlhydra fed various numbers of shrimps per day. I cell-free hydra digest food poorly(the shrimps forced into the hydra gastric cavity appear nearly intact after a day ofdigestion) so that we cannot tell how much food the hydra are actually digesting. Their
/ cell-free hydra 25
budding rates (o-2-o-6 buds/hydra/day) resemble those of normal hydra fed between2 and 8 shrimps per day.
The morphological budding stages of I cell-free hydra are nearly normal, as definedin Otto & Campbell (1977).
Table 5. Budding rates of I cell-free and normal hydra
Type of hydra
Feedinglevel,
shrimps/day
No. ofhydra
No. ofbuds
Timeobserved,
daysBudding rate,
buds/hydra/day
I cell-freeI cell-freeI cell-freeNormalNormalNormalNormalNormal
888i248
25
3179
2424242424
20
5°46
o176572
145
II
141266666
o-6iO-2I
o-43o0 1 2
o-45050I-OI
1 2 3 4 5 6Time of regeneration, days
Fig. 3. Regeneration of I cell-free hydra ( • — • ) and of control hydra starved for1 (O O), S (° °), and 10 days ( # - - - # ) before experimentation. Ordinateshows the regenerated percentage of the initial tentacle number in each group of10 animals. The original tentacle numbers were 9-6, 5-9, 5-6 and 6-i tentacles/polyp inthe 4 groups, respectively. Thus the I cell-free hydra actually regenerated a highernumber of tentacles than the control hydra.
Regeneration and polarity. I cell-free hydra regenerate. Fig. 3 illustrates the rates ofregeneration by I cell-free hydra and control hydra decapitated in the upper columnregion. Regeneration of I cell-free hydra is slightly retarded at first but catches upwith controls. It is not markedly different from controls
Midgastric regions of hydra were marked in the distal part by a non-diffusable,vital carbon technique (Campbell, 1973) and then isolated. We scored whether
26 B. A. Marcum and R. D. Campbell
hydranth regeneration occurred at the proximal or at the distal end. In all cases I cell-free tissue preserved its polarity (Table 6).
Polarity reversal. I cell-free hydra show nearly normal kinetics of polarity reversal.Elsewhere (Marcum, Campbell & Romero, 1977) we report experiments that demon-strate this. These experiments show not only that I cell-free tissue can reverse itspolarity normally, but also that I cell-free hydranth and peduncle tissue is capable ofinducing polarity reversal in intervening tissue.
Table 6. Polarity of regeneration of I cell-free gastric segments
Experiment
i
2
3
Table 7.
Donor
No. ofhydra
619
8
No. of regenerationsA
Withretained polarity
6198
Induction of secondary axis by tissue
Host No.
5
Withreversed polarity
000
implants
Type of inductionA
Positive Negative
Hypostomal tissue implantsI cell-free I cell-freeI cell-free NormalNormal I cell-freeNormal Normal
Gastric region tissue implantsI cell-free I cell-freeI cell-free NormalNormal I cell-freeNormal Normal
oo1
o
9943
Induction. Normal hypostomal (mouth) tissue, if implanted into the body columnof another hydra, will induce a secondary axis to form in the host column, whiletissue taken from the gastric region will not (Browne, 1909). We tested the inductiveproperties, as well as the susceptibility to induction, of I cell-free tissue by implantingsmall pieces into both I cell-free and normal hydra columns. Similar grafts were madeusing tissue taken from normal hydra. Table 7 shows the frequency with whichinduction occurred. In general, hypostomal tissue implanted into a column induced asecondary axis, regardless of whether the donor and/or the host was I cell-free ornormal. Conversely, gastric regions did not induce, regardless of the animals used.Only 3 grafts (lines 3 and 7) out of 50 yielded different results. Thus I cell-free tissueis as capable of, and susceptible to, induction as is normal tissue.
Determination. When the hydranth is removed from a hydra, the distal-most re-maining tissue regenerates a new hydranth. Webster & Wolpert (1966) have shown that
I cell-free hydra 27
such regenerating tissue first (in about 6 h) becomes irrevocably determined to formhydranth, and only later (in about 2 days) does it actively differentiate. Determinationcan be assayed by implanting tissue into the side of a normal host column: if theimplant is determined to form hydranth it will organize a secondary axis on the host;if the tissue is not determined it will simply be resbrbed.
Table 8 compares the inductive capacity of subhypostomal tissue of I cell-free andof normal hydra. Freshly isolated subhypostomal tissue, from either type of hydra, hasno inductive capacity upon grafting into a host (lines 1 and 3). Subhypostomal tissueallowed to regenerate for 6 h does have moderate inductive capacity (lines 2 and 4).Thus I cell-free tissue can undergo hypostomal determination (Webster & Wolpert,1966) similar to normal tissue.
Table 8. Determination of regenerating subhypostomal tissue
Source ofsubhypostomal tissue
Time allowedto regenerate,
h.No. ofcases
% of casesshowing
determination
I cell-free hydraI cell-free hydraNormal hydraNormal hydra
11
427
o64o
23
1002 3 4 5 6 7 8 9 1 0 1 1 1 2
Time, days
2 3 4 5 6 7 8 9 1011
Time, days
Fig. 4. Tissue movements in I cell-free hydra. Left: one polyp, whose appearance isdrawn in the centre, was vitally marked with 9 spots on day o. On successive days thepositions of each spot is recorded. The first (uppermost) spot moved to the tip of a ten-tacle and disappeared on day 11; the seventh moved on to a bud on day 6; the lower-most moved off the base on day 12. The budding region is shown by the bracket to theleft of the polyp. Right: composite of 86 marker movements on 9 hydra. Positions ofall marks which began in each tenth of the body column were averaged on successivedays. This method underestimates movement rates because the most rapidly movingspots frequently left the animal through buds and hence did not contribute to laterposition averages. Bracket to right of polyp shows budding positions.
28 B. A. Marcum and R. D. Campbell
Tissue movements. We have studied tissue movements in 9 I cell-free hydra byvitally marking a number of spots along the columns and watching these marks moveduring the course of 2 weeks. Fig. 4 (left) illustrates the pattern of marker movementin one I cell-free hydra. Tissue moves upwards into the tentacles and downwards intothe buds and basal disk. This gives rise to a stationary region in the upper region ofthe column where tissue does not move relative to the morphology. Tentacle turnoverrequires about 10 days. These features are typical of normal hydra tissue movements(Campbell, 1967). Fig. 4 (right) shows the composite data for all 9 hydra studied.
DISCUSSION
Colchicine treatment offers a method for completely eliminating interstitial, nemato-blast, nematocyte, gland and nerve cells from hydra, leaving the remaining depletedepithelia in the form of viable hydra. This is the first such method available. Someprevious methods have achieved partial success. X-irradiation and nitrogen-mustard(Strelin, 1929; Brien & Reniers-Decoen, 1955; Burnett & Diehl, 1964; Tardent &Morgenthaler, 1966) have been used to eliminate nerve and interstitial cells, but theremaining hydra invariably died; since nematocysts were also eliminated the hydracould not eat, but also the epithelial cells were probably extensively damaged by theirradiation. No detailed analyses of cell population levels following X-irradiation ornitrogen-mustard treatments have been published. It is reported that methylene blue(Diehl & Burnett, 1964) and eserine sulphate (Bursztajn, 1974) can be used to elimi-nate nerve cells, but quantitative data regarding effectiveness are not available, and thetreatments are not permanent because interstitial cells quickly redifferentiate intonerves. In an accompanying article Sugiyama & Fujisawa (1977) show that I cell-freehydra can be obtained through genetic methods.
Hydra once rendered free of interstitial cells subsequently remain free of inter-stitial cells (currently we have maintained clones of I cell-free hydra for over 16months). Thus interstitial cells are not readily produced through dedifferentiation ofepithelial cells, a plausible transformation which has been frequently suggested in theliterature. However, we have on a few occasions seen a few interstitial cells in hydrain which we expected to find none (for example, see Table 3, clone IIK, count madeon 21/4/76). Such instances could be explained by an infrequent (1 in perhaps io6
cell cycles) transformation of epithelial cells into interstitial cells. Alternatively, theseisolated encounters of interstitial cells could also be explained by contamination ofour maceration fluid or stocks, or by a low number of insterstitial cells persisting butusually escaping detection.
Three unexpected characters of colchicine-treated hydra are very favourable to theexperimenter. The first is that the hydra are viable. The second is that in animals inwhich a low level of interstitial cells persists, these few interstitial cells do not pro-liferate to repopulate the hydra. Rather low numbers of interstitial cells remain stable.For example, clone 46-1 has about 1 % of the normal numbers of interstitial cells andhas maintained this low level for more than a year of active culturing (see Table 4).These polyps each contain only one or two score of interstitial cells; what feedback
/ cell-free hydra 29
controls enable such low population densities to be maintained is an intriguingquestion. The third unexpected character of treated hydra is that when only a fewinterstitial cells remain, as in clone 46-1, there are no derivative cells to be found.Thus animals which have a few interstitial cells are still free of nematocytes, gland andnerve cells, so the complete elimination of interstitial cells may not be crucial for allexperiments on nerve-free (or nematocyte-, gland-, or gamete-free) hydra tissue.There is apparently a threshold in interstitial cell density, below which they do notdifferentiate into derivative cells.
I cell-free hydra exhibit little behaviour and have a high threshold to stimulation,consistent with the view that nerve cells do have pacemaker and sensory functions inhydra. However, nerve-free hydra tissue can respond to strong stimuli, contract andconduct induced excitation (Campbell, Josephson, Schwab & Rushforth, 1976;Schwab et al. 19766).
The growth and morphogenetic capacities of I cell-free hydra are extensive. Thisshows that epithelial cells are capable of carrying out most or all morphogeneticactivities of hydra. This may be surprising in view of accumulating evidence that thenervous system plays critical roles in hydra (and other animals) development. Thefollowing evidence, although none of it decisive, at least points toward nerve cellspatterning morphogenesis through neurosecretory functions. Some hydra cells havethe cytological appearance of neurosecretory cells and discharge their vesicles pre-paratory to polyp regeneration (Lentz, 1965; Davis, 1973). Extracts of hydra enrichedin nerve cell materials affect hydra development; low molecular weight substanceswith inhibitory and stimulating activity have been partly purified and characterized(Lesh, 1970; Schaller & Gierer, 1973; Berking, 1974; Schaller, 1973, 1975, 19760,6).Nerve cells are most abundant in those hydra regions (hypostome, bud tip and basaldisk) which have inducing capacity and which are developmentally dominant (Bodeet al. 1973; Davis, 1973). Packets of nerve cells differentiate locally during initiationof buds and regeneration (Bode et al. 1973). Elimination of nerves with eserinesulphate abolishes regenerative ability (Bursztajn, 1974). Finally, theoretical models ofhydra development have been proposed which are based in part on nerve cell proper-ties (Burnett, 1966; Gierer & Meinhardt, 1972; MacWilliams, Kafatos & Bossert,1970) (see also Bursztajn & Davis, 1974; Davis & Bursztajn, 1974).
Therefore it is surprising that nerve-free hydra display a broad spectrum ofdevelopmental capacities: growth, tissue displacement, budding, regeneration, main-tenance and reversal of tissue polarity and induction. We have found 4 alternativeways to reconcile our observations with those of the literature:
(1) Nerve cells may not be involved in hydra development after all. This is the moststraightforward way to interpret our results, but the 3 other possibilities meritconsideration.
(2) Nerve cells may play a role in 'fine tuning' developmental patterns which arebasically established by the epithelial cells. This interpretation is suggested by the factthat I cell-free hydra are slightly abnormal. For example, they have tentacles, buttentacle disposition is slightly irregular. During tissue repolarization, intermediateforms of regeneration are not seen as often as in normal regeneration (Marcum et al.
3 CEL 29
30 B. A. Marcum and R. D. Campbell
1977). It is also true that the demonstrated developmental effects of factors extractedfrom hydra are quantitative rather than qualitative. For example, addition of a stimula-tory factor, extracted from hydra, to regenerating hydra statistically promotes a fewpercent increase in tentacle number (Schaller, 1973) or changes the duration of themitotic cycle (Schaller, 19766), but ordinarily does not alter qualitatively the patternsof development.
(3) Nerve cells play essential roles in patterning hydra, but in their absence compensa-tory activities can be displayed by the epithelial cells. This interpretation is suggestedby the observation that it takes a few days for colchicine-treated hydra to re-establisha normal morphology (Campbell, 1976), indicating that time might be required for acompensatory function to arise. Precedent for this interpretation comes from amphibianlimbs. Limb regeneration is dependent upon nerves. Yet aneurogenic limbs (thosearising on denervated embryos) (Singer, 1974) and limbs subjected to the prolongedabsence of nerves (Thornton, 1970) are able to regenerate without neurotrophicstimulation but lose this capability if reinnervated (Thornton & Thornton, 1970).Thus apparently essential nerve activities are compensated for by the remaining celltypes.
(4) Nerve cells and epithelial cells may both be exerting identical or overlapping controlsover hydra development. The idea of redundancy in developmental controls has beenlargely neglected but is consistent with observations on more complex developmentalphenomena such as canalization (Waddington, 1940) and induction (Jacobson, 1966).
By studying experimentally assembled chimeric hydra, as described below, thesevarious alternatives should be clearly distinguishable. Whatever the normal develop-mental interrelations between nerve and epithelial cells, the I cell-free hydra are oneof the most complex developing systems known consisting of simple apparentlyhomogeneous epithelia.
We consider that a major importance of I cell-free hydra is the spectrum of newexperimentation which they suddenly make possible. For example, the followingtypes of study can now be undertaken:
Selective repopulation of I cell-free hydra by particular cell types. For example, agraft of normal tissue will introduce nematocytes to the I cell-free tentacles within afew hours, a day or more before the tentacles acquire nerves; thus one can study therole of nerves in nematocyst discharge. Unusual repopulated ratios or levels of cellsof the interstitial cell lineage could be produced to complement other methods (Bode,Flick & Smith, 1976) of examining what regulates the development of this cell line.
Chimera formation, by repopulating depleted hydra of one strain or species withinterstitial cells of another. Such chimeras will reveal which cell types normallycontrol development and morphology of hydra (Sugiyama & Fujisawa, 1977).
Observation of immobilized tissue, to visualize long-term or slow developmentalprocesses. Since I cell-free hydra are motionless, it is possible to compress the entirebudding process into a short time-lapse movie, and should be possible to view nema-tocyte and other cell movements. The propagated annulations described in Resultsrepresent one class of process which is completely masked by contractile movementsof normal hydra.
/ cell-free hydra 31
Observations on simplified tissues, since the ectoderm of I cell-free hydra is so muchsimplified from that of normal hydra. For example, in I cell-free hydra it is possibleto visualize the epithelio-muscular cell processes using polarization microscopy (Otto,1977)-
We thank Nancy Wanek for help in these experiments. This investigation was supportedby grant number NS 12446 and Postdoctoral Training Grant number HD 07029, awarded bythe National Institutes of Health.
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(Received 10 May 1977)
