J. Cell Set. 29t 17-33(1978)
Printed in Great Britain © Company of Biologists Limited 1978
17
DEVELOPMENT OF HYDRA LACKING NERVE
AND INTERSTITIAL CELLS
BEVERLY A. MARCUM* AND RICHARD D. CAMPBELL
Department 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 derivatives by colchicine treatment. These hydra were then cloned and cultivated for 18 months and
their developmental capacities were studied. Some experimental hydra possessed a few (about
1 % of the normal numbers) interstitial cells and retained this low level during prolonged
culture and active growth without the differentiation of I-cells into specialized cells. Other
hydra were completely freed of interstitial cells by the colchicine treatment. Maceration and
histological analyses showed that once a hydra is freed of all interstitial cells it does not recover
them, nor do its buds contain interstitial cells. I cell-free hydra also lack nerve cells, nematocytes, gametes and endodermal gland cells, and the tissue consists solely of ectodermal and
endodermal epithelial cells. Hydra completely lacking interstitial cells grow, bud, exhibit
tissue renewal patterns, regenerate and preserve polarity generally typical of normal hydra.
I cell-free hypostomal tissue has inductive capacity, as does normal hypostomal tissue, when
implanted in I cell-free or normal gastric tissue. Regenerating I cell-free tissue undergoes
precocious determination as does normal tissue. Only in some quantitative aspects do I cellfree hydra develop abnormally. We conclude that hydra consisting only of epithelial cells are
capable of essentially normal development.
INTRODUCTION
Hydra tissues contain just a few cell types. Diverse current investigations are attempting 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 the
major classes of cells, the interstitial cell and its derivatives, and testing the developmental 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 of
the nerve cells, nematocytes and gametes (David & Gierer, 1974). These differentiated
and specialized cells do not themselves divide. Therefore, by eliminating the interstitial cells, one can effectively and permanently prevent the formation of nerve cells
and other interstitial cell products.
In this paper we describe a method for eliminating interstitial cells and their derivatives, making it possible to study the development and physiology of hydra which are
• Address for correspondence: Dr Beverly Marcum, Center for Pathobiology, University
of 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 hydra
development (Tardent, 1954; Lentz, 1966; Schaller, 1973). We also report here that
hydra 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 in
Lake Zurich in 1962. This is the same hydra population that has been used extensively in other
researches (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 species
of hydra.
Colchicine (obtained from Sigma Corp., and kept under refrigeration as powder) treatment
consisted 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 were
washed repeatedly and washing was repeated several times daily thereafter for 1 week. The
same animals were retreated with colchicine after 10-20 days. The entire regime described here
will be termed a 'double colchicine treatment.' All subsequent culturing was done in the same
M solution containing 50 fig/ml rifampicin. Except for a 3-day recovery period following each
treatment, the hydra were fed daily. Feeding was accomplished by inserting freshly killed
Artemia nauplii through the hydra's mouth, using a finely drawn polyethylene mouth pipette
to 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 and
12 h after a meal each gastric cavity is well flushed with a jet of culture solution by means of a
polyethylene micropipette inserted through the hydra's mouth. The hydra do not survive
without this treatment.
Maceration and cell identification followed the procedures of David (1973). Interstitial cell
repopulation was accomplished by grafting a top or bottom half of a vitally marked, normal
hydra to the complementary piece of a colchicine-treated polyp. Several days later the normal
tissue was cut away using the vital marking as a guide. Vital marking was carried out by the
methods of Campbell (1973), using small spots of injected ink for studying tissue movements
and large spots, extending around the circumference of the hydra, in experiments involving
grafting. 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 the
epithelial cells. Fig. 1 illustrates the time course of relative cell abundance changes in
response to colchicine treatment. Interstitial cells, nematoblasts and nematocytes
become depleted within a few days after each colchicine treatment. Nerve and gland
cells decline in numbers somewhat more slowly. These kinetics are highly reproducible with this strain of hydra (compare with fig. 4 in Campbell, 1976).
After 2 weeks following the second colchicine treatment, few non-epithelial cells are
present and the cell population levels do not show extensive further changes. More
extensive cell counts (Table 1) indicate that hydra sampled at this time contain a
low average number of non-epithelial cells and that this number varies somewhat
from one preparation to the next.
From a population of twice-treated hydra we cloned 13 individuals (methods for
feeding and culturing these hydra are described under Materials and methods). When
each clone consisted of several hydra we began to analyse the cell compositions of
/ cell-free hydra
60
8
^12
16
20
24
r
28
Days after first colchicine treatment
Fig. 1. Time course of cell disappearance following colchicine treatment. The
population levels of nerve cells ( • — • ) , interstitial cells (O
O ) and endodermal
gland cells ( x
x ) are expressed relative to epithelial cell number. Colchicine
treatments (0-4 %, 8 h) were given on days o and 11 (arrows).
Table 1. Number of nerve and interstitial cells present 2 weeks after
the second colchicine treatment
•Experiment
no.
No. of
hydra
1
2
3
12
Total
3
No. of cells
sampled
No. of
nerve cells
No. of
interstitial
cells
1500
o
o
o
9
6000
12
5
27
19500
5
17
Each experiment involved independent colchicine treatment.
12000
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 2
lacked interstitial cells and interstitial cell derivatives.
Three of these clones were then propagated for over a year, and more extensive cell
counts (Table 3) were made at intervals during this period.
Apparently 2 of these clones (IE and IIA) are completely free of all cell types
except 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 interstitial
cell 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 structures
looked like nerve or interstitial cells, and we have not been able to prepare macerations
without them. These bodies may represent degenerating cell fragments arising from
Table 2. Relative cell compositions of polyps in 13 clones of colchicine-treated hydra
Cell counts
Gland
Unrecognizable
0
34
0
0
40
3
0
0
62
1
0
0
0
6
952
0
0
0
95O
952
0
0
0
0
0
0
43
48
56
46
II L
HID
IIIE
961
0
0
0
0
0
0
39
48
1
952
959
0
0
0
39
2
IK
875
656
2
0
0
55
0
0
0
41
4
3
943
948
2
0
0
55
0
1
0
0
51
0
973
949
3
4
0
0
0
2
0
0
954
0
0
0
44
44
74
45
0
0
95O
962
1
2
0
0
0
0
46
36
0
900
2
0
0
98
0
Clone
label
IE
IIA
II K
IL
IIC
HE
IIIA
IIIB
IIIL
Big
interstitial
Nerve
Nematocyte
lineage
0
0
0
0
0
95i
Epithelial
966
957
1171
1352
0
0
2
1
3
2
1
3
Each line represents a separate polyp. Cell types are labelled according to David (1973)
except: 'Nematocyte lineage' includes all little interstitial cells, nematoblasts and nematocytes; ' gland' includes all endodermal glandular cells and ectodermal basal disk epithelial
cells; 'unrecognizable' includes structures resembling cells but not fitting into the other
categories.
endodermal cell sloughing at the basal disk and tentacle tips, and are visible in histological sections. However, since it is conceivable that they are aberrant nerve or interstitial cells, and unrecognizable, this debris puts a limit on cell determination by the
maceration 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 not
all interstitial cells and interstitial cell derivatives. Those which do remain, however,
are not equally distributed among hydra. Some hydra entirely lack interstitial, nematoblast, nematocytes and nerve cells, and these hydra can be selected and cloned as
/ cell-free hydra
21
Table 3. Relative cell compositions of 3 clones of hydra treated twice
with colchicine
Cell counts
Clone
IE
IIA
UK
ControlJ
Nematocyte
lineage
Epithelial
Big
interstitial
962
0
0
0
0
0
0
34
43
4
956
Nerve
Gland
UnrecogDate #
nizable day, month, year
1
971
0
0
0
29
0
936
0
0
0
58
925
0
0
0
72
6
3
974
1909
979
1989
0
0
0
10
16
0
0
0
81
10
0
0
0
24
0
0
0
0
0
11
10
990
0
0
0
0
2712
0
0
0
0
9
1151
951
949
0
0
0
1
0
0
0
62
43
0
0
0
51
0
900
0
0
0
7
95O
0
0
0
93
47
966
0
0
0
1935
0
0
0
32
62
4568
0
0
0
0
992
0
0
0
0
2993
0
0
0
95°
956
958
0
0
0
0
0
952
952
0
979
2934
978
2686
259
318
(29%)
6
3
2
3
42
5/i i/75
26/11/75
2/1/76
3/i/76
3/1/76
23/1/76
18/3/76
21/4/76
i9/8/ 7 6t
23/8/76
14/4/77
29/10/75
6/11/75
26/11/75
20/12/75
23/12/75
13/3/76
20/4/76
i9/8/76t
0
8
7
23/8/76
3/3/77
0
5°
0
0
43
1
0
0
42
0
0
0
0
0
0
48
48
0
0
0
0
21
0
2
0
0
64
0
0
0
0
0
22
0
0
0
0
14
6/1i/75
26/11/75
30/12/75
1/1/76
1/1/76
18/3/76
21/4/76
23/8/76
3/3/77
242
43
37
39O
401
66
58
(4%)
(4O%)
(6-2 %)
186
(21 %)
0
See Table 2 for explanation of column headings. After 21/4/76 ' Gland' refers only to endodermal gland cells.
• Colchicine treatment was begun on 23/9/75.
t IE and I I A 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. Marcum and R. D. Campbell
polyps consisting only of epithelial cells. Once hydra are free of interstitial cells, they
remain free of interstitial cells through extensive growth, budding and culturing over
a period of many months.
Histological evidence that some clones lack interstitial cells
To confirm our maceration counts we prepared cloned colchicine-treated hydra for
histological examination. Two animals from each of 3 clones (IE, IIA and IIK)
were serially sectioned transversely and each cell identified and counted. No interstitial cells, nerve cells, or cells of the nematocyte lineage were found. The details
of 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. These
have been produced by either single colchicine treatment or by partial repopulation of
colchicine-treated hydra through temporary implant of normal tissue. Table 4 illusTable 4. Relative cell composition of hydra in clone no. 46-1 *, containing relatively
few interstitial cells
Cell counts
Nerve
Nematocyte
lineage
17
17
13
0
0
80
2
0
0
10
10/5/76
0
0
23
0
8
5
8
0
0
0
2
0
0
0
6
29/7/76
2/8/76
3/8/76
13
18
0
0
1
0
25/10/76
Big
Epithelial
1216
969
979
993
985
2731
1982
interstitial
Gland
UnrecogDate,
nizable day, month, year
4/"/7S
5/2/77
See Table 2 for explanation of column headings. After 10/5/76 'Gland' refers only to endodermal gland cells.
• The original polyp of this clone was a twice-treated individual repopulated with a small
number of interstitial cells from a temporary graft of normal hydra tissue on 3/10/75.
0
0
0
0
trates 3 unusual characters of such polyps, as exemplified by our clone number
46-1. First, interstitial cells do not increase in number and thus repopulate the hydra.
(At interstitial cell levels of greater than several percent of normal, repopulation
occurs.) Second, no nerve, gland, or nematocyte cells are found in these hydra. When
only a small number of interstitial cells remain in an animal, these cells do not differentiate. Third, hydra containing low levels of interstitial cells have the morphological
appearance of hydra which are completely free of interstitial cells.
Thus polyps retaining a few interstitial cells can be propagated indefinitely in a
condition 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 twice
with colchicine and subsequently propagated asexually, and which are apparently
completely free of interstitial, nematoblast, nematocyte, endodermal gland and
nerve cells. These hydra consist of only epithelial cells. Therefore, the term ' I cellfree' is a convenient label for a condition broader than simply lacking interstitial
cells.
I cell-free hydra grow if fed. When buds detach and reach full size they cannot be
distinguished from the parents. Three clones (I E, IIA and III E) have been extensively
cultured for over a year and have produced more than 500 polyps. However, this
number does not represent the maximum possible number of hydra obtainable since
many polyps were used for experiments, Thus, these hydra are capable of extensive
and probably unlimited growth, as are normal hydra (Brien & Reniers-Decoen, 1949).
I cell-free hydra were repopulated with normal interstitial cells by means of a
temporary tissue implant (see Materials and methods). Repopulated hydra appear
normal in morphology, growth, budding and behaviour. Since no new epithelial cells
are introduced by this repopulation procedure, we conclude that the epithelial cell
of 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 months
of culturing. Initially they are less swollen and more coloured than later. Initially they
have greater digestive capacities, as evidence by extensive breakdown of the shrimp
which are pressed into their gastric columns. After a few months, shrimp are unchanged
in appearance after remaining in the gastric cavity for a day or longer. After prolonged
culture, I cell-free hydra tissue becomes less resistant to stretching and less coloured by
food. 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 body
proportions are atypical.
Fig. 2 shows normal and I cell-free hydra. I cell-free hydra have swollen distal
columns, narrow peduncles and small basal disks. The basal disks are sticky and accumulate white envelopes, presumably through secretory activity, but the animals do
not attach to the substrate. Buds remain perched on parent stalks for many days. The
lower part of the body is frequently annulated. The tissue is nearly colourless, transparent and stretched. The hypostome is large, flat and thin rather than being a
thick conical protrusion as in normal hydra. I cell-free hydra have tentacles that are
more numerous than normal and are often of different lengths. Tentacles may be
irregularly spaced but remain more or less in a whorl. The tentacles are also shorter
than normal and are straight and slender. The morphology of I cell-free hydra
varies with feeding. In one set of hydra fed regularly the tentacle number averaged
9-6, 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 not
flushed following feeding) the hydra may quickly shrink and die; the first sign of
suboptimal 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 over
a year without interstitial cells or derivatives, A, normal Hydra attenuata; B, I cell-free
hydra; c, I cell-free hydra starved for 10 days, showing extra hypostome; D, I cell-free
hydra 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 activity
that can be observed directly. They do not show any response to reduced glutathione
added to the culture solution (W. Heagy, personal communication).
Some spontaneous activity can be observed in time-lapse movies of I cell-free
hydra. Once or several times a day the fluid in the gastric cavity discharges suddenly
into the culture and the hydra collapses. Re-extension follows. Slight column
pulsing and tentacle bending at their junction with the body occurs at irregular
intervals. Annulations on the column propagate upwards along the column. All of
these 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 the
hydra to such stimulation, but it is much less sensitive than that of a normal hydra.
Further behavioural and electrophysiological characteristics of colchicine-treated
hydra 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 control
hydra 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 of
digestion) 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 between
2 and 8 shrimps per day.
The morphological budding stages of I cell-free hydra are nearly normal, as defined
in Otto & Campbell (1977).
Table 5. Budding rates of I cell-free and normal hydra
Feeding
level,
shrimps/
No. of
hydra
No. of
buds
Type of hydra
day
I cell-free
I cell-free
I cell-free
Normal
Normal
Normal
Normal
Normal
8
8
8
3
17
i
2
24
24
24
24
65
72
145
4
8
25
1
Time
observed,
days
Budding rate,
buds/hydra/day
20
II
o-6i
14
12
O-2I
9
5°
46
24
o
6
6
6
6
6
17
2
3
4
5
Time of regeneration, days
o-43
o
012
o-45
050
I-OI
6
Fig. 3. Regeneration of I cell-free hydra ( • — • ) and of control hydra starved for
1 (O
O), S (°
°), and 10 days ( # - - - # ) before experimentation. Ordinate
shows the regenerated percentage of the initial tentacle number in each group of
10 animals. The original tentacle numbers were 9-6, 5-9, 5-6 and 6-i tentacles/polyp in
the 4 groups, respectively. Thus the I cell-free hydra actually regenerated a higher
number of tentacles than the control hydra.
Regeneration and polarity. I cell-free hydra regenerate. Fig. 3 illustrates the rates of
regeneration by I cell-free hydra and control hydra decapitated in the upper column
region. Regeneration of I cell-free hydra is slightly retarded at first but catches up
with 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 cellfree 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 demonstrate this. These experiments show not only that I cell-free tissue can reverse its
polarity normally, but also that I cell-free hydranth and peduncle tissue is capable of
inducing polarity reversal in intervening tissue.
Table 6. Polarity of regeneration of I cell-free gastric segments
No. of regenerations5
A
Experiment
No. of
hydra
6
i
2
19
3
8
With
retained polarity
With
reversed polarity
6
0
0
0
19
8
Table 7. Induction of secondary axis by tissue implants
Type of induction
A
Donor
Host
No.
Positive
Negative
o
o
9
9
4
3
Hypostomal tissue implants
I cell-free
I cell-free
I cell-free
Normal
Normal
I cell-free
Normal
Normal
Gastric region tissue implants
I cell-free
I cell-free
I cell-free
Normal
Normal
I cell-free
Normal
Normal
1
o
Induction. Normal hypostomal (mouth) tissue, if implanted into the body column
of another hydra, will induce a secondary axis to form in the host column, while
tissue taken from the gastric region will not (Browne, 1909). We tested the inductive
properties, as well as the susceptibility to induction, of I cell-free tissue by implanting
small pieces into both I cell-free and normal hydra columns. Similar grafts were made
using tissue taken from normal hydra. Table 7 shows the frequency with which
induction occurred. In general, hypostomal tissue implanted into a column induced a
secondary axis, regardless of whether the donor and/or the host was I cell-free or
normal. 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 tissue
is as capable of, and susceptible to, induction as is normal tissue.
Determination. When the hydranth is removed from a hydra, the distal-most remaining 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 form
hydranth, and only later (in about 2 days) does it actively differentiate. Determination
can be assayed by implanting tissue into the side of a normal host column: if the
implant 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 and
of normal hydra. Freshly isolated subhypostomal tissue, from either type of hydra, has
no inductive capacity upon grafting into a host (lines 1 and 3). Subhypostomal tissue
allowed 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 of
subhypostomal tissue
Time allowed
to regenerate,
h.
I cell-free hydra
I cell-free hydra
Normal hydra
Normal hydra
No. of
cases
% of cases
showing
determination
11
42
7
o
64
o
23
100
2 3 4 5 6 7 8 9 1 0 1 1 1 2
Time, days
2 3 4 5 6 7 8 9
Time, days
1011
Fig. 4. Tissue movements in I cell-free hydra. Left: one polyp, whose appearance is
drawn in the centre, was vitally marked with 9 spots on day o. On successive days the
positions of each spot is recorded. The first (uppermost) spot moved to the tip of a tentacle and disappeared on day 11; the seventh moved on to a bud on day 6; the lowermost moved off the base on day 12. The budding region is shown by the bracket to the
left of the polyp. Right: composite of 86 marker movements on 9 hydra. Positions of
all marks which began in each tenth of the body column were averaged on successive
days. This method underestimates movement rates because the most rapidly moving
spots frequently left the animal through buds and hence did not contribute to later
position 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 by
vitally marking a number of spots along the columns and watching these marks move
during the course of 2 weeks. Fig. 4 (left) illustrates the pattern of marker movement
in one I cell-free hydra. Tissue moves upwards into the tentacles and downwards into
the buds and basal disk. This gives rise to a stationary region in the upper region of
the column where tissue does not move relative to the morphology. Tentacle turnover
requires 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, nematoblast, nematocyte, gland and nerve cells from hydra, leaving the remaining depleted
epithelia in the form of viable hydra. This is the first such method available. Some
previous 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 the
remaining hydra invariably died; since nematocysts were also eliminated the hydra
could not eat, but also the epithelial cells were probably extensively damaged by the
irradiation. No detailed analyses of cell population levels following X-irradiation or
nitrogen-mustard treatments have been published. It is reported that methylene blue
(Diehl & Burnett, 1964) and eserine sulphate (Bursztajn, 1974) can be used to eliminate nerve cells, but quantitative data regarding effectiveness are not available, and the
treatments are not permanent because interstitial cells quickly redifferentiate into
nerves. In an accompanying article Sugiyama & Fujisawa (1977) show that I cell-free
hydra can be obtained through genetic methods.
Hydra once rendered free of interstitial cells subsequently remain free of interstitial cells (currently we have maintained clones of I cell-free hydra for over 16
months). Thus interstitial cells are not readily produced through dedifferentiation of
epithelial cells, a plausible transformation which has been frequently suggested in the
literature. However, we have on a few occasions seen a few interstitial cells in hydra
in which we expected to find none (for example, see Table 3, clone IIK, count made
on 21/4/76). Such instances could be explained by an infrequent (1 in perhaps io 6
cell cycles) transformation of epithelial cells into interstitial cells. Alternatively, these
isolated encounters of interstitial cells could also be explained by contamination of
our maceration fluid or stocks, or by a low number of insterstitial cells persisting but
usually escaping detection.
Three unexpected characters of colchicine-treated hydra are very favourable to the
experimenter. The first is that the hydra are viable. The second is that in animals in
which a low level of interstitial cells persists, these few interstitial cells do not proliferate 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 and
has 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 intriguing
question. The third unexpected character of treated hydra is that when only a few
interstitial 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 and
nerve cells, so the complete elimination of interstitial cells may not be crucial for all
experiments on nerve-free (or nematocyte-, gland-, or gamete-free) hydra tissue.
There is apparently a threshold in interstitial cell density, below which they do not
differentiate 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 in
hydra. However, nerve-free hydra tissue can respond to strong stimuli, contract and
conduct induced excitation (Campbell, Josephson, Schwab & Rushforth, 1976;
Schwab et al. 19766).
The growth and morphogenetic capacities of I cell-free hydra are extensive. This
shows that epithelial cells are capable of carrying out most or all morphogenetic
activities of hydra. This may be surprising in view of accumulating evidence that the
nervous system plays critical roles in hydra (and other animals) development. The
following evidence, although none of it decisive, at least points toward nerve cells
patterning morphogenesis through neurosecretory functions. Some hydra cells have
the cytological appearance of neurosecretory cells and discharge their vesicles preparatory to polyp regeneration (Lentz, 1965; Davis, 1973). Extracts of hydra enriched
in nerve cell materials affect hydra development; low molecular weight substances
with 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 basal
disk) which have inducing capacity and which are developmentally dominant (Bode
et al. 1973; Davis, 1973). Packets of nerve cells differentiate locally during initiation
of buds and regeneration (Bode et al. 1973). Elimination of nerves with eserine
sulphate abolishes regenerative ability (Bursztajn, 1974). Finally, theoretical models of
hydra development have been proposed which are based in part on nerve cell properties (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 of
developmental capacities: growth, tissue displacement, budding, regeneration, maintenance and reversal of tissue polarity and induction. We have found 4 alternative
ways to reconcile our observations with those of the literature:
(1) Nerve cells may not be involved in hydra development after all. This is the most
straightforward way to interpret our results, but the 3 other possibilities merit
consideration.
(2) Nerve cells may play a role in 'fine tuning' developmental patterns which are
basically established by the epithelial cells. This interpretation is suggested by the fact
that I cell-free hydra are slightly abnormal. For example, they have tentacles, but
tentacle disposition is slightly irregular. During tissue repolarization, intermediate
forms 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 extracted
from hydra are quantitative rather than qualitative. For example, addition of a stimulatory factor, extracted from hydra, to regenerating hydra statistically promotes a few
percent increase in tentacle number (Schaller, 1973) or changes the duration of the
mitotic cycle (Schaller, 19766), but ordinarily does not alter qualitatively the patterns
of development.
(3) Nerve cells play essential roles in patterning hydra, but in their absence compensatory activities can be displayed by the epithelial cells. This interpretation is suggested
by the observation that it takes a few days for colchicine-treated hydra to re-establish
a normal morphology (Campbell, 1976), indicating that time might be required for a
compensatory function to arise. Precedent for this interpretation comes from amphibian
limbs. Limb regeneration is dependent upon nerves. Yet aneurogenic limbs (those
arising on denervated embryos) (Singer, 1974) and limbs subjected to the prolonged
absence of nerves (Thornton, 1970) are able to regenerate without neurotrophic
stimulation but lose this capability if reinnervated (Thornton & Thornton, 1970).
Thus apparently essential nerve activities are compensated for by the remaining cell
types.
(4) Nerve cells and epithelial cells may both be exerting identical or overlapping controls
over hydra development. The idea of redundancy in developmental controls has been
largely neglected but is consistent with observations on more complex developmental
phenomena such as canalization (Waddington, 1940) and induction (Jacobson, 1966).
By studying experimentally assembled chimeric hydra, as described below, these
various alternatives should be clearly distinguishable. Whatever the normal developmental interrelations between nerve and epithelial cells, the I cell-free hydra are one
of the most complex developing systems known consisting of simple apparently
homogeneous epithelia.
We consider that a major importance of I cell-free hydra is the spectrum of new
experimentation which they suddenly make possible. For example, the following
types of study can now be undertaken:
Selective repopulation of I cell-free hydra by particular cell types. For example, a
graft of normal tissue will introduce nematocytes to the I cell-free tentacles within a
few hours, a day or more before the tentacles acquire nerves; thus one can study the
role of nerves in nematocyst discharge. Unusual repopulated ratios or levels of cells
of 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 with
interstitial cells of another. Such chimeras will reveal which cell types normally
control development and morphology of hydra (Sugiyama & Fujisawa, 1977).
Observation of immobilized tissue, to visualize long-term or slow developmental
processes. Since I cell-free hydra are motionless, it is possible to compress the entire
budding process into a short time-lapse movie, and should be possible to view nematocyte and other cell movements. The propagated annulations described in Results
represent one class of process which is completely masked by contractile movements
of normal hydra.
/ cell-free hydra
31
Observations on simplified tissues, since the ectoderm of I cell-free hydra is so much
simplified from that of normal hydra. For example, in I cell-free hydra it is possible
to visualize the epithelio-muscular cell processes using polarization microscopy (Otto,
1977)We thank Nancy Wanek for help in these experiments. This investigation was supported
by grant number NS 12446 and Postdoctoral Training Grant number H D 07029, awarded by
the National Institutes of Health.
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