AMFR. ZOOL., 14:543-550
(1974).
Activity of Hydra Cells in vitro and in Regenerating Cell Reaggregates
HANS R. BODE
Max Planck Institut fiir Virusforschimg, 74 Tubingen, West Germany, and
University of California, Irvine, California 92664
SYNOPSIS. Hydra were disaggregated into single cells and maintained in a complex medium for a few days. The cells are capable of macromolecular synthesis, of processing
RXA, and of carrying out the formation of a nematocyst capsule. Single C2l!s can lie
centrifuged into aggregates which will develop into normal hydra. Changes in morphology and cell composition are described. Interstitial cells after a 1-day delay resume
their normal mitotic and differentiation activity in the aggregates. The method provides new means for studying problems of cell differentiation in hydra.
The ability to culture cells in vitro has
obvious advantages for developmental studies, especially cell differentiation. Culture
of coelenterate cells is still in a very primitive state, although some success has been
reported for marine coelenterates. Martin
and Tardent (1963) were able to maintain explants of Tubularia tissue in vitro
for several weeks. Cells that appeared to
be early stage nematoblasts migrated from
the explant and later began to proliferate
and differentiate. Similarly Burnett et al.
(1968) reported that several cell types
migrating from Tubularia explants were
capable of cell division and differentiation. The only true cell culture has been
achieved by Phillips (1961). Starting with
cell suspensions he was able to establish
cultures of cells of the sea anemone,
Anthoplenra elegantissima. Cultures of several cell lines that he isolated were transferred 20 to 30 times and kept under in
vitro cultivation for more than a year.
Unfortunately, the culture of hydra cells
has so far been unsuccessful, though several attempts have been made (Papenfuss
and Bokenham, 1939; Sanyal and Mookerjee, 1960; Cerame-Vivas, 1961; Li et al.,
1963). One reason why culture of hydra
cells has been very difficult is that the
obvious starting point used for developing
a culture medium for almost all other
animal cells is not available in hydra.
Most cells are isosmotic with the fluids
that surround them such as blood or, in
the case of marine coelenterates, sea water.
Hydra has no intercellular fluid to speak
of. The osmolarity of the cytoplasm, 120
mosmols., though difficult to measure accurately, is much higher than that of either
the external medium, 7 mosmols., or the
fluid in the gastral cavity, 60 mosmols.
(Benos and Prusch, 1972). Thus, there are
no clear reference points for either the
correct osmolarity or salt composition for
a balanced salt solution, let alone the
problem of nutrients.
As part of a study of the differentiation
of the multipotent interstitial cell in hydra
undertaken by a group of people working
with Gierer at the Max Planck Institut fiir
Virusforschimg in Tiibingen, West Germany, Trenkner and I attempted to culture cells of H. attenuata in vitro. The
results of these efforts, which have been
described more fully elsewhere (Trenkner
et al., 1973), will be presented as well as
a new and useful approach to the study
of cell differentiation in hydra (Gierer
et al., 1972) which grew out of these
efforts.
ACTIVITY OF HYDRA CELLS IN VITRO
In part the author's research was supported by
the National Science Foundation Grant GB 29284.
Hydra can be disaggregated into single
viable cells either by soaking animals in
543
544
HANS R.
BODE
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FIG. 1. Sucrose gradient profiles of RNA pulselabelled with H3-uridine in intact animals (A) and
cultured cells (B) . The radioactive RXA was extracted from intact animals 45 min (top) , 2.5 hr,
and 20.5 hr (bottom) after injection. RXA was
extracted from cultured cells 2.5 hr (top) , 5 hr,
and 23 hr (bottom) after addition of H3-uridine.
(0
0 ) H3-uridine labeled RXA. (O
O)
unlabeled bulk hydra RXA. (Modified from Trenkner et al, 1973.)
ACTIVITY OF HYDRA CELLS IN VITRO
a balanced salt solution for 30 min and
then gently disrupting the tissue with a
Pasteur pipette, or by stirring pieces in
balanced salt solution with low concentrations of trypsin. In either case cells are
obtained that can be maintained for a
few days in a complex medium consisting
of salts, amino acids, vitamins, sugars, and
bovine serum albumin. The ion concentration was chosen to yield an osmolarity of
210 mosmols. This value was obtained both
empirically and from calculations based
on the tissue volume and the dry weight
of the various ions per hydra.
Though the cells do not stick to the
substrate and cell division has not been
observed, they are capable of macromolecular synthesis. They will synthesize DNA,
RNA, and protein as measured by incorporation of radioactive precursors into
acid-insoluble material. The radioactivity
is rendered acid-soluble by treatment with
the appropriate enzyme: DNase, RNase,
or pronase.
Further, the cells are apparently capable
of processing RNA molecules in vitro as
they do in the intact animal. This was
shown by comparing the fate of RNA that
had been pulse-labelled with H3-uridine
in vitro with that pulse-labelled in the
animal. If H3-uridine is injected into the
gastral cavity, more than 90% of the radioactivity that is taken up is in acid-insoluble
material within 2 hr (David, unpublished) . This procedure amounts to a 2-hr
pulse. Animals were injected with H3-uridine and the RNA extracted after 45 min,
2.5 hr and 20.5 hr, and sedimented in
sucrose gradients using nonradioactive bulk
RNA as a reference. As shown in Figure
\A, a large fraction of the H3-uridine
material sediments rapidly at first with
sedimentation coefficients greater than the
28 S ribosomal RNA component. A 2.5. hr
discrete ribosomal RNA peaks appear, and
by 20.5 hr the labelled material has the
same distribution as the bulk of the unlabelled material. The same experiment
was carried out in vitro by exposing the
cells to H3-uridine for 2.5 hr and then further incubating them in non-radioactive
545
uridine (Fig. \B). A similar trend is apparent. After 2.5 hr there is a large peak
greater than 28 S which declines at 5 hr
and disappears by 23 hr. Concurrently
rRNA begins to appear at 5 hr and is very
much pronounced at 23 hr. In both experiments nearly all the radioactive material
is degraded by RNase treatment. Thus, the
size, distribution, and processing of RNA
synthesized in cultured cells is similar to
that in the animal.
Beyond the capacity of the cells to synthesize macromolecules, their ability to differentiate in vitro was examined. For this
the interstitial cell —» nematocyte pathway
was a suitable first choice because: (i)
nematocytes are made in large numbers so
that a fairly large percentage of all cells
are either nematocytes or intermediates;
(ii) the nematocyst capsule can be recognized quite early during differentiation as
a clear vesicle. This is useful in case only
a limited amount of differentiation takes
place.
The particular differentiation step examined was the transition from the little
interstitial cell, the intermediate capable
of cell division (see David, 1973, for terminology) , to the developing nematoblast,
the cell with a distinct vesicle or later an
identifiable type of nematocyst. Because
the little I-cells are capable of DNA synthesis whereas nematoblasts are not, the
transition can be followed with H3-thymidine. H3-thymidine injected into the animal appears in little I-cells immediately,
and in nematoblasts only 24 to 36 hr
later (David, unpublished). To determine
whether the transition can take place in
vitro, cells in culture were evposed to
H3-thymidine, and then examined autoradiographically 4 hr and 36 hr later. No
labelled nematoblasts were found at either
time indicating the transition did not take
place. In a second experiment, animals
were labelled with H3-thymidine, disaggregated 10 hr later, and maintained in culture medium. Labelled cells were analyzed
autoradiographically immediately after disaggregation and after 36 hr in vitro. No
labelled nematoblasts were found in ani-
546
HANS R. BODE
ACTIVITY OF HYDRA CELLS IN VITRO
mals which were autoradiographed immediately after disaggregation. After 36 hr
in culture, about 20% of the labelled small
cells were clearly nematoblasts. This is
similar to the percentage of labeled nematoblasts found in vivo after 36 hr (Bode,
unpublished; David, personal communication) . They contained a very prominent
normal appearing nematocyst somewhere
between immature and complete in its
development. This indicates the cells are
capable of carrying out the synthesis of
the nematocyst capsule in vitro.
The different results in the two experiments indicate that a necessary step that
took place in the animal in the 10 hr after
injection of H3-thymidine and before disaggregation was not carried out in vitro.
One possibility is that the cells cannot
undergo a necessary cell division in vitro.
The G-2 period of these rapidly dividing
little I-cells is about 4 hr (David and
Campbell, personal communication). Thus,
those little I-cells that are labelled could
readily undergo cell division in the 10
hr before the animal is disaggregated.
These cells could continue the differentiation into nematoblasts in vitro.
In summary, hydra cells can be maintained in vitro for a few days. They are
capable of synthesizing macromolecules,
processing RNA, and carrying out the formation of a nematocyst capsule.
ACTIVITY OF CELLS IX REGENERATING
CELL AGGREGATES
Though cell culture does not exist yet
for hydra cells, another method has been
developed which has many of the desirable
attributes of cell culture for the study of
cell differentiation (Gierer et al., 1972).
The method is as follows. Disaggregated
cells of H. attenuata, instead of being
placed in culture medium, are gently cenFIG. 2. Stages in the development of hydra from
an aggregate of cells. A, Rounded-off clump of
150,000 cells (~H/; hydra). The clump diameter
is 0.5 mm (6 hr). B, Hollow sphere (20 to 30 hr).
C, Appearance of tentacle buds (40 to 48 hr). D,
Appearance of hypostomes (60 to 80 hr). E, Well-
517
trifuged into clumps of cells. The clumps
are placed in a 70 mosmol. balanced salt
solution, and over a period of 20 to 30 hr
the solution is diluted stepwise to hydra
medium. The clump of cells will develop
into one or more hydra depending on the
number of cells in the initial clump.
The various stages of the process are
shown in Figure 2. Initially, the solid
clump of cells is irregular in shape and
has a bumpy surface of round cells randomly stuck together. Within 4 to 6 hr
(Fig. 2A), the clump has rounded into
a sphere. The cells on the surface have
flattened and formed a smooth coherent
sheet. After 20 to 30 hr the sphere has
become hollow (Fig. 2.B). Many cells have
been expelled as indicated by the mass of
debris outside the aggregate. The hollow
sphere consists of two concentric layers
which resemble the ectodermal layer on
the outside and the pigmented gastroderm
on the inside. A new mesoglea is visible
between the tissue layers sometime between
24 and 36 hr. By 40 to 48 hr (Fig. 2C)
tentacle buds begin to appear, usually
spread irregularly over the surface. Then,
at 21/2 to 3 days, hypos tomes appear (Fig.
2D). Tentacles around a hypostome develop normally as part of a head region.
Other tentacles disappear, and may be resorbed. By 4 to 6 days (Figs. 2E,F) these
monsters are capable of ingesting Artemia.
Eventually the several hypostomes will
divide up the tissue and normal animals
develop. Later these animals are capable
of budding.
There are variations on this sequence of
events. Aggregates made of cells of Chlorohydra viridissima will form hypostomes
first, and then develop tentacles around
the hypostome, or the two events are apt
to be simultaneous. The same holds true
for aggregates made of cells from the gastric
region alone of H. attenuata. Also, aggredeveloped head regions and body columns (6 days).
F, A developing aggregate made of two or three
times more cells than the first aggregate (10 days) .
The times refer to the time elapsed after aggregation. Each stage is magnified 45 X.
548
HANS R. BODE
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20
40
60
80
100180
time after aggregation (h)
FIG. 3. Cell composition of developing aggregates.
The cell composition of three to five aggregates
was quantitated with the maceration technique
(David, 1973) at each time shown. In such preparations all hydra cell types can be recognized and
counted. For each of the cell types analyzed, the
data are presented as the number of cells of the
cell type/total cells of the aggregate expressed as a
percentage for each time. The individual curves are
in A: nerve cell
(0
0 ) ; gland cell
# ) ; big
(O
O) : i n B- epithelial cell ( #
interstitial cell (Q
O) • little-interstitial cell
(^
^ ) ; nematoblast (A
A) . The total
cells/aggregate at each time are in B ( •
•) •
(Data from Gierer et al., 1972.)
ACTIVITY OF HYDRA CELLS IN VITRO
gates of basal disk cells alone will form
basal disks first and may or may not form
hypostomes later.
The changes in cell number and cell
composition accompanying the morphological changes of the developing aggregates
were also examined. The most dramatic
event was the loss of l<uge numbers of
cells during the transformation of a solid
clump of cells into a hollow sphere. As
shown in Figure 3, a clump starting with
150,000 cells has been reduced to 50,000
cells in 2 days. These losses are not distributed evenly over the several cell types
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FIG. 4. Mitolic index, number of big interstitial
cells in nests, and number of nerve cells during
aggregate development. The counts were done on
the same maceration preparations used for Figure
3. The cell numbers in li are expressed per aggregate. A, Mitotic index of big I-cells. B, Number of
big I-cclls in nests of two (Q
Q) and nests
of four (%
%); ( O — * O ) > number of n m e
cells. (From Gierer et al., 1972.)
549
but are quite selective. At the time the
clumps are formed, the composition is
similar to that of the normal animal (Bode
et al., 1973). During the first 2 days, the
percentage of big interstitial cells/aggregate remains unchanged. This indicates
they are lost at the same rate as the total
ioss of ceiis from the aggregate (see Fig. 4).
The same is true for gland cells. In contrast, epithelial cells are preferentially retained while little J-cells and nematoblasts
are selectively lost. Thereafter, the cell
number/aggregate slowly begins to rise,
and again the increases are selective. At
first little 1-cells begin to rise in number
and later nematoblasts. Nerve cells are
selectively lost during the first 24 hr, and
then recover to the normal level by 48 hr.
Because the differentiation activity of
the big interstitial cell is of particular interest, its behavior was examied in detail.
During the first 18 hr (Fig. 4/4), the
mitotic index was around 0.5% which is
considerably less than the 3% found in
the intact tissue (Bode, unpublished; David
and Campbell, personal communication).
After the first day the mitotic index begins
to rise, and by 2 days has regained the
normal level. Early in aggregate development big I-cells are found as single cells
which is in contrast to the clusters of two
and four that are prevalent in normal tissue. As mitotic activity recovers the number of pairs also rises (Fig. 4/J) and a day
later the clusters of four begin to reappear. Still later nests of 8 and 16 little
I-cells are found, followed by nests of
nematoblasts, as has already been mentioned. Similarly a day after mitotic activity of the I-cells recovers, the numbers of
nerve cells begin to rise (Fig. 4JS) suggesting I-cell division is necessary before
nerve cell differentiation can occur. These
results indicate that the conditions within
the aggregate permit the interstitial cell to
resume its normal mitotic and differentiation behavior within a day or so.
The differentiation of interstitial cells
into nerves in aggregates was also examined directly. Animals were labelled
with H3-thymidine, immediately disaggregated after labelling, the cells added to
550
HANS R. BODE
suspensions of unlabelled cells, aggregates
formed, and periodically the type of labelled cells analyzed with autoradiography.
In such animals the interstitial cells, but
not the nerves, are labelled. In the normal
animal, labelled nerve cells will appear
24 hr later. In the aggregates no labelled
nerve cells were found after 24 hr but at
48 hr about 16% of them were labelled
while about 25% of the interstitial cells
were labelled. This indicates that somewhat more than half the nerve cells found
at 48 hr in the developing aggregates arose
by I-cell differentiation, and probably accounts for the marked rise in the nerve
cell population at that time.
In summary, the aggregation technique
provides a new approach to the study of
cell differentiation in hydra. In a manner
similar to cell culture, H3-thymidinelabelled cells can be treated in a number
of ways in vitro such as by exposure to
drugs or possible differentiation signals.
Then, instead of following their fate in
vitro, the treated cells are added to the
aggregates and their behavior analyzed.
This is true for either populations of cells
or single cells obtained by micromanipulation. Further, separation of cell types will
provide another variable that can be manipulated. Then, relatively pure populations of cells can be treated and analyzed,
and aggregates can be made with various
cell compositions. At present partial separation of cell types has been achieved.
Thus, it seems unnecessary at present to
pursue the development of a cell culture,
for many questions concerning interstitial
cell differentiation can now be attacked
with this method.
Finally, other than cell differentiation,
the method provides a measure of manipulation for morphogenetic studies in hydra
that has not previously existed. Gierer et
al. (1972) have already exploited it for a
study on tissue polarity in hydra.
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Bocle, H., S. Berking, C. N. David, A. Gierer, H.
Schaller, and E. Trenkner. 1973. Quantitative
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Buriiclt, A. L., F. E. Ruffing, J. Zongkcr, and A.
Necco. 1968. Growth and differentiation of
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Cerame-Vivas, M. J. 1961. Separation of cell layers
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tissue culture of coelenterate cell lines, p. 245254. In H. M. Lenhoff and W. F. Loomis [ed.],
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