/ . Embryol. exp. Morph. Vol. 29, l,pp. 1-13,1973
Printed in Great Britain
]
In vitro studies in Tubularia: a morphological
role for digestive cells
By MARC H. GLICKMAN 1 AND GEORGIA E. LESH-LAURIE
From the Department of Biology, Case Western Reserve University,
Cleveland, Ohio
SUMMARY
A chemically defined in vitro system for Tubularia has been developed. At 24 h after
explantation of coenosarc, digestive cells attached to the substrate and migrated from the
explant. The digestive cells migrated by a gliding motion with a fan-like membrane acting as a
leading edge. Within 48 h a digestive cell monolayer was formed and invasion of this area by
epithelio-muscular, gland and interstitial cells occurred. Autoradiographic study of 48-72 h
cultures treated with [3H]thymidine showed nuclear incorporation of the label in digestive,
epithelio-muscular, interstitial, cnidoblast and gland cells.
When explants were grown on collagen-coated coverslips, accelerated attachment and
migration of digestive cells was observed. Explants were also grown on Millipore filters. No
digestive cell attachment occurred but epithelio-muscular, gland and interstitial cell attachments to the filter were observed. From these experiments, a morphological role for the
digestive cells as a substrate for other cells of the coenosarc is postulated.
Hydranth extract was supplemented to the culture medium. Studies with this material were
performed with coenosarc from 'late summer' animals in which only 10-15 % of the explants
normally entered culture. However, with the addition of the extract, 100 % of the explants
went into culture. Interstitial cell populations increased 2-3 times in extract-treated explants.
INTRODUCTION
Cnidarians have often provided the experimental system for studying the
morphological role of cell layers, as well as control mechanisms involved in the
expression of a differentiated state (Tardent, 1963). Previous studies, however,
have been restricted by a single limitation, the absence of in vitro investigations.
This limitation has resulted in incomplete descriptions of cellular migration in
these organisms, the morphological role(s) of the cell layers, and the
differentiation sequence of the pluripotent interstitial cells.
Extended maintenance of cnidarian cells in a totally defined medium has not
been previously reported. Sanyal & Mookerjee (1960) studied Hydra cells in
vitro using pond water as a culture medium. The cells, however, lived only a
few hours. Phillips (1961) using edamine, an enzymic digest of lactalbumin,
succeeded in keeping Anemone cells viable for at least 12 days. In this system,
however, all the cells appeared morphologically identical.
1
Author's address: Department of Biology, Case Western Reserve University, Cleveland,
Ohio 44106, U.S.A.
I
E M B 29
2
M. H. GLICKMAN AND G. E. LESH-LAURIE
Martin & Tardent (1963), employing 'slow-to-coagulate' lobster blood,
successfully cultured Tubularia cells. The major drawbacks to this study were
the complex culture methodology and the use of lobster blood. Since no serum
is found in situ in cnidarians, investigators of cellular differentiation or the
morphological role of cell layers normally desire a medium without serum.
An additional advantage to a medium lacking serum is the opportunity to
develop a system to test chemical parameters of differentiation directly, without
the possibility of serum interacting with the normal differentiation process.
Burnett, Ruffing, Zongker & Necco (1968) employed a chemically defined
medium for Tubularia cells. They observed mitotic figures in stained preparations
and what appeared to be differentiation in interstitial cells. Unfortunately, to
date, these studies have not been successfully repeated or extended.
In this study a chemically defined in vitro system for Tubularia cells has been
developed. Time-lapse microcinematography has been used to detail the initial
establishment of cultures, cellular migration and cell differentiation. From
analyses of these films and direct light-microscope observations, a morphological role for gastrodermal digestive cells as a substrate for other cell types
has been hypothesized.
MATERIALS AND METHODS
Tubularia spectabilis were routinely cultured at Instant Ocean (10) (Aquarium
Systems, Inc., Wickliffe, Ohio, U.S.A.) at 12 °C. They were fed Artemia salina
nauplii twice weekly and cleaned 4 h after feeding. All experimental animals
were starved 24 h prior to use during which time they were placed in a beaker
of 10 containing 0-25 % kanamycin.
In vitro procedures
Animals were cut 2 cm from the hydranth and the excised section was placed
in filtered 10 which had been titrated to pH 3 with HC1. This process relaxed
the animals and allowed the coenosarc to remain intact. The hydranth was then
excised and discarded. The proximal end of the hydrocaulus was held with
forceps while coenosarc was extruded from the perisarc with a second pair of
forceps. Isolated coenosarc was transferred to a sterile physiological salt
solution known as Necco's solution (Burnett et al. 1968). This solution consisted
of (g/1): NaCl, 25-800; KC1,0-644; CaCl2,0-644; MgCl 2 .6H 2 O, 3-080; KH 2 PO 4 ,
0020; Na 2 HPO 4 , 0080; NaHCO 3 , 0-200; Na2SO4, 0-920; pyruvic acid, 1-500;
fumaric acid, 1-050; L glutamine, 1-090; phenol red, 0-020.
Using cataract knives, the cylindrical coenosarc was cut longitudinally
yielding a flat sheet of cells. This flat sheet of cells was then transferred to a
Petri dish which contained the final culture medium. This medium consisted
of 3 parts Necco's solution, 1 part glass distilled H2O, 0-4 parts Eagle's MEM
(Grand Island Biological Comp.)
Digestive cells in Tubularia
3
The following antibiotics were added to the final culture medium: 0-3 g/1
kanamycin, 0-25 g/1 penicillin, 0-25 g/1 streptomycin, 0-25 g/1 fumagillin B.
After 5 min in the Petri dish the coenosarc was transferred into fresh final
medium on a substrate. Substrates used in this study included coverslips,
autoclaved 0-22 /im Millipore filters, 1 jum Nucleopore filter and collagen (rat
tail) coated coverslips. After the coenosarc was maneuvered to lay flat on the
substrate, excess liquid was withdrawn, leaving only a small amount of fluid
surrounding the cells. The substrate with the explant was placed in a Petri dish
and the Petri dish placed in a desiccator. All cultures were incubated at 12 °C.
After the initial 3 days, fresh medium was added to the cultures. New medium
was then added every 2 days. Change of medium was accomplished by
pipetting fresh medium, maintained at 12 °C, on to the explant for 15 min and
then withdrawing the excess liquid.
The final culture medium was aliquoted and stored at — 20 °C. All procedures
were carried out under sterile conditions. All solutions were filtered through
3 GS sintered glass filters.
Microcinema tographic obser va tions
A Bolex camera with a model 500 Sage instrument panel was used to observe
the cells in culture. A Zeiss photomicroscope was employed for observing the
cultures. Cinematographic observations were recorded at 4 frames/min on
Kodak Tri X reversal film.
A u toradiography
Coverslip cultures were incubated in media containing 25 /tCi/ml [3H]thymidine (6-7 c/ml) for 24 h. These cultures were washed two times for 10 min
each with cold thymidine solution (concentration ten times that of labeled
solution) and fixed 24 h later for autoradiography. All cultures were fixed in
Bouin's fixative. The coverslip cultures were dipped in a mixture of equal parts
liquefied gel form nuclear track emulsion (Ilford K-5) and distilled water,
allowed to dry, and placed in light tight boxes with a desiccant for 3-4 weeks at
2 °C. After the incubation period the coverslips were developed in Kodak D-19
and fixed with Kodak Rapid fix. After water washing the cultures were stained
with 0-01 % aqueous toluidine blue (pH 8), dehydrated, cleared in toluene and
mounted. The cellular incorporation of [3H]thymidine was compared in three
regions of the cultures. All labeled nuclei in a 1 mm2 area of five randomly
chosen fields on each of 11 explants were counted. The labeled nuclei were then
separated into the regions where they were found. These regions consisted of
the central explant; the periphery of the explant, an area the width of four cells;
and the monolayer of digestive cells, which extended beyond the periphery of
the explant.
4
M. H. GLICKMAN AND G. E. LESH-LAURIE
RESULTS
Initially, criteria were established to determine the time at which Tubularia
cells had successfully gone into culture. The principal criteria included the
attachment of gastrodermal digestive cells to the substrate, cellular migration
and the absence of discharged nematocysts.
While preparing and testing over 3500 cultures, it was also observed that
cultures had to be maintained at 11-12 °C. When the temperature exceeded
15 °C, gastrodermal digestive cells often retracted from the substrate. In this
investigation cultures were maintained for only 4-week periods. During the
initial testing period, however, cultures were maintained for up to 8 weeks.
24 h after explantation
At 24 h after explantation on to coverslips, large pigment-filled (carotenoid)
gastrodermal digestive cells (Cohen, 1952) attached to the substrate. These
cells also retained attachments to the explant. The cells displayed a fibroblastlike appearance during the attachment process. Furthermore, a large fanshaped extension was exhibited at their point of attachment to the substrate
(Fig. 1). Cinematographic evidence revealed that this extension ruffled. The
fan-like membrane occurred not only at the free edge of the gastrodermal
digestive cells but also extended around the sides of the cells (Fig. 2). Attachment was not overtly polarized, yet a radial outgrowth of the digestive cells was
often observed.
48 h after explantation
Between 24-48 h of culture, the digestive cells became vacuolized. During
this time period time-lapse photography revealed two migration patterns in the
FIGURES
1-5
Fig. 1. Twenty-four hours after explantation, gastrodermal digestive cells (D)
attached to the substrate by fan-shaped membranes at arrows. Note digestive cells
that have individually migrated from explant. Phase contrast, x 90.
Fig. 2. Attachment by digestive cells (D) to substrate by fan-shaped membrane and
cytoplasmic processes noted by arrows. Phase contrast, x 90.
Fig. 3. Forty-eight hours after explantation, gastrodermal digestive cell monolayer
is formed; nuclei (n) of digestive cells are indicated. Attachments to substrate are
indicated by arrows. Phase contrast, x 90.
Fig. 4. Sixty hours after explantation, the gastrodermal monolayer is invaded by
gland cells (g) and epidermal cells (e). Note vacuolated gastrodermal cells. Toluidine
blue, x 132.
Fig. 5. (A) Autoradiographic study of 48-72 h culture showing [3H]thymidine incorporation in interstitial cell (/) and digestive cell nucleus (Dn). Note unlabeled
nematocyst (ne). x 800.
(B) Autoradiogram showing [3H]thymidine incorporation in digestive cell nucleus
(Dn). x800.
Digestive cells in Tubularia
5B
M. H. GLICKMAN AND G. E. LESH-LAURIE
Table 1. Labeled nuclei in one representative 48-72 h culture
(No. of labeled nuclei in 1 mm2 area of tissue culture.)
Central Periphery of Digestive cell
explant
explant
monolayer Total
60
28
18
16
32
68
37
22
24
53
15
16
12
10
12
143
81
52
50
97
digestive cells. Initially, the digestive cells migrated individually or retained a
thin membranous attachment to the tissue explant. Cinematographic evidence
showed that these cells migrated by a gliding motion. When individually
migrating digestive cells came in contact with each other, they did not pile up
but formed a sheet or monolayer of digestive cells. Once a monolayer was
formed, the digestive cells moved as a sheet of cells with the most peripheral
cells exhibiting a fan-shaped membrane (Fig. 3). The fan-shaped membranes of
these peripheral digestive cells acted as a leading edge and appeared to pull the
other cells of the monolayer. These membranes also appeared to be the main
source of attachment of the sheet to the coverslip for when these membranes
detached from the surface, the entire sheet retracted to the explant.
60 h after explantation and established cultures
At 60 h after explanting, time-lapse studies showed the monolayer of gastrodermal digestive cells was infiltrated by gland cells, nematocysts, epitheliomuscular cells and interstitial cells (Fig. 4). Gastrodermal digestive cell migration
still occurred but at a reduced rate. As epithelio-muscular cells and gland cells
FIGURES
6-8
Fig. 6. (A) Forty-eight hours explant grown on Millipore filter showing cell attachments from the explant to the filter. Note interstitial cell (/) attachment. Toluidine
blue. x75.
(B) High magnification of the area indicated in (A) showing interstitial cell (/),
gland cell (g) and epithelio-muscular cell (e) attachments. Toluidine blue, x 800.
Fig. 7. (A) Twenty hours after coenosarc is explanted on collagen-coated coverslip.
Gastrodermal digestive cell (D) monolayer has been established. Phase contrast.
xl60.
(B) Forty-eight hours after coenosarc is explanted on a collagen-coated coverslip.
Same age culture as Fig. 3. Note attachment process (a) of gastrodermal cells and
infiltration of monolayer by gland cell (g) and epidermal cell (e). Toluidine blue.
xl32.
Fig. 8. (A), (B) Forty-eight to sixty hours after cultures have been treated with
hydranth extract. Note numerous interstitial cell nests (/), cnidoblasts (en), nerves
(ne) and nematocysts (nm) on digestive cell monolayer (Dn). Toluidine blue, x 320.
Digestive cells in Tubularia
8A *
8
M. H. GLICKMAN AND G. E. LESH-LAURIE
migrated on to the monolayer of gastrodermal cells, the digestive cells underwent a process of vacuolation leaving areas of what seemed to be extremely thin
cytoplasm. This event was observed consistently and may be essential for further
development of the culture.
From time-lapse observations, interstitial cells appeared to extend small
filopodial processes as they migrated. These cells moved either between digestive
cell attachments or on the monolayer of the vacuolated digestive cells.
Nematocysts per se did not migrate actively over the monolayer but were
passively pushed by the migratory movements of the epithelio-muscular cells.
Gland cells were observed to migrate by an amoeboid motion between
vacuolated digestive cell attachments.
Autoradiographic studies of 48-72 h cultures treated with [3H]thymidine
showed nuclear incorporation of label in digestive cells, epithelio-muscular
cells, interstitial cells, cnidoblasts and gland cells (Fig. 5 A, B). Approximately
84 % of the total nuclear incorporation occurred in the explant; 16 % of the
incorporation occurred in the monolayer. Approximately 58 % of the nuclear
incorporation in the explant was concentrated at the periphery of the explant
(Table 1). These observations are consistent with the suggestion that these cells
were undergoing mitosis. Although no grain counts were made, heavy nuclear
incorporation allowed relative ease in judging differences in amounts of
[3H]thymidine incorporation. Reduced amounts of silver grains in interstitial
cells and digestive cells in the monolayer portion of the tissue culture were
observed.
Substrate studies
Various substrates were employed to test their effect on the establishment and
outgrowth of cultures. Tissues were routinely explanted on to glass coverslips.
These cultures were therefore used as a standard for substrate comparisons.
Explants were cultured on 0-22 jum Millipore filters and 1 /im. Nucleopore
filters (Table 2). Explants on neither substrate exhibited digestive cell attachments. Forty-eight hours after explantation it appeared that gland, interstitial
and epithelio-muscular cells migrated directly on to the filter and attached to
it (Fig. 6 A, B).
Explants were also placed on collagen-coated coverslips (Fig. 7 A, B). Within
12 h digestive cells attached to the substrate. Twelve hours later the migrated
area was invaded by gland, epithelio-muscular and interstitial cells. Therefore,
the rate of digestive cell attachment occurred twice as rapidly on collagen-coated
coverslips as it did on uncoated glass coverslips. Also, a greater concentration
of digestive cells per unit area migrated from the explant on to the collagencoated coverslips. Finally, migration and attachment of vacuolated digestive
cells occurred farther away from the explants than in control cultures. Migration
and outgrowth of gland, epithelio-muscular and interstitial cells also occurred
twice as rapidly in the collagen substrate cultures (Table 2).
Collagen
22 /tm Millipore; 1 /tm Nucleopore
filter
Glass coverslip: normal tissue
' Late summer' tissue
'Late summer' tissue and
hydranth extract
'Late summer' tissue 24 and
48 h in culture and hydranth
extract
Substrate
24/24-^8
24/24-48
12/12-24
24/24-48
(after addition
of extract)
12/12-24
—
80
10-15
100
100
98
30
2000
55
60
40
250
50
Explants
that become
cultures/50
Digestive cell
Number of
attachment/migration
explants
cultures
(h)
(%)
—
4-5
24-30
24-48
(on to filter)
30
3-2
2-2
1-65
48
48
60
60
Invasion of monolayer
Distance of
by epithelio-muscular,
digestive cell
interstitial and gland
from periphery of
cells
explant (mm)
(h)
Table 2. Substrate studies of'm vitro experiments
p"
H
E?
<§
b
10
M. H. GLICKMAN AND G. E. LESH-LAURIE
Effects of hydranth extract on tissue culture
A labile material has been isolated from hydranth extracts of Tubularia. This
material presumably is derived from nerve cells found in the hydranth region
(Brooks, Lesh-Laurie & Glickman, 1971). Preliminary tissue culture studies
with this material were performed with 'late summer' animals. Only 10-15 %
of the explants from coenosarc of 'late summer' animals normally entered
culture (Table 2). After 24 and 48 h these cultures normally showed reduced
amounts of digestive cell outgrowth. When the culture medium was supplemented with the hydranth extract (1 part extract :70 parts medium), 100 %
of the explants went into culture. In addition digestive cells attached to the
substrate within 12 h after explantation. Attachment and migration of the
digestive cells therefore occurred at twice the rate found in 'normal' coverslip
cultures (i.e. employing coenosarc from any other time of the year) (Table 2).
At no time did bacterial contamination occur or increase with the addition
of the hydranth extract. Also, no osmotic swelling of cells was observed.
Several unique effects on cell populations were observed between 48-60 h
culture after using the hydranth extract as a culture supplement. Interstitial
cell nest population increased 2-3 times. The relative frequency of interstitial
cell derivatives (e.g. nerves, cnidoblasts and nematocysts) also increased
(Fig. 7A, B).
This isolated material has also been added to 'late summer' explants 24 and
48 h after the coenosarc was placed in culture. Before adding the isolated material,
these explants displayed none of the criteria for successful cultures. Twenty-four
hours after the addition of the isolated material, 100 % of these explants showed
digestive cell attachment and migration. The normal sequence of tissue culture
events then followed.
DISCUSSION
A chemically defined in vitro system has been established for Tubularia.
Twenty-four hours after explantation, attachment, migration and outgrowth of
digestive cells occur. In 48-60 h cultures, interstitial cells, epithelio-muscular
cells and gland cells migrate on to the monolayer of gastrodermal digestive
cells. All cells in vitro bear considerable similarity cytologically to the cells in
situ. From the results of this investigation it is postulated that the elongate,
vacuolated, digestive cells play an important role in the establishment and
maintenance of cnidarian tissue cultures. This role may involve the digestive
cells' migratory activity and their capacity to serve as a substrate for other cells.
Digestive cells: morphological role
The digestive cells were the first cells to attach to the substrate and migrate
farthest in culture. When grown on glass coverslips, the digestive cells attached
to the substratum by a large fan-like membrane, similar to that observed in
Digestive cells in Tubularia
11
vertebrate embryonic cells (Abercrombie, 1965). Time-lapse revealed that this
fan-like membrane acted as a leading edge for the gliding movement of the
cells. The digestive cells moved either individually or in sheets. Digestive cells
moving individually did not pile up when coming into contact with other
digestive cells but formed a monolayer of cells. This behavior is analogous to
"contact inhibition' described by Abercrombie (1967).
Explants grown on collagen-coated coverslips exhibited a more rapid attachment and accelerated migration. The effects observed with collagen-coated
coverslips indicated that the digestive cells adhered more quickly to collagen
than to glass. An hypothesis explaining this increased rate of attachment and
migration could center around collagen acting as a mesogleal mimic. Burnett &
Hausman (1969) offered a theory that digestive cells and epidermal cells use
the collagen-elastin mesoglea as a point of attachment for migratory activity.
In vitro observations are consistent with this suggestion.
Time-lapse and cytological observations revealed that epithelio-muscular
cells, interstitial cells and gland cells normally wandered only over a digestive
cell monolayer. When the explanted tissue was placed on a substrate (e.g. a
Millipore filter) interstitial cells, gland cells and epithelio-muscular cells were
found several hundred micra away from the initial tissue mass. No digestive
cell monolayer attached nor formed in the presence of the niters. These
observations indicate that a smooth or collagen-like substrate may be necessary
for digestive cell attachment. Interstitial, gland and epithelio-muscular cells may
require a substratum with grooves; a substratum that may be similar to a
digestive cell monolayer. The depressions and/or grooves may serve as
anchoring points for the cells' processes.
The morphological importance of digestive cells in situ can be postulated from
the results of this in vitro investigation. In situ, when a hydranth is excised, the
mesoglea in the animal breaks down (Tardent, 1962). At the wound surface,
digestive cells are the first cells to close the wound (Glickman, unpublished
observation). Epidermal cells then migrate over the digestive cells. Therefore,
all of the in situ evidence supports the hypothesis that digestive cells serve as a
substrate for other cells similar to that which occurs in vitro. Without a suitable
substrate (e.g. the surface of digestive cells) epithelio-muscular cells may be
incapable of migratory movement. Tardent & Erymann (1959) showed that
during hydranth histogenesis, digestive cells shifted distally while the epitheliomuscular cells remained attached to the perisarc. From their reports one could
postulate that it is only after digestive cells have been established as a substrate
that the other cell types may then migrate into the primordial region.
Nuclear activity
Autoradiographic studies using [3H]thymidine have shown nuclear incorporation of the label in digestive cells, epithelio-muscular cells, interstitial cells,
gland cells and cnidoblasts. Fifty-eight per cent of the nuclear incorporation in
12
M. H. GLICKMAN AND G. E. LESH-LAURIE
the explant occurred in cells at the periphery. This observation is consistent
with the idea that the periphery of the explant may act as a 'feeder' for the
migrated area.
In situ, Tardent (1963) reported that the number of mitotic figures in regenerating and non-regenerating Tubularia was extremely low. Campbell (1967)
found mitotic activity in the hydranth region but proximal to the hydranth, few
mitoses were observed. The in vitro system may exhibit an increase in the rate
of mitotic activity as compared to the in situ situation. However, detailed
quantitative analyses must be performed in order to compare both systems
accurately.
Effects of hydranth extract
Without the perisarc which is normally impermeable to drugs (Burnett et al.
1968), cells in an in vitro system can be treated directly to note cellular effects
from any isolatable hydranth materials. Burnett (1966) has hypothesized that a
quantitative change in an inducer may result in a qualitative effect on cnidarian
cellular differentiation. Lesh (1970) showed that in regenerating Hydra, the
relative frequency of interstitial cells and interstitial cell derivatives varied with
varying concentrations of an isolated inductive material. Since interstitial cells
are present in tubularian tissue cultures, it may be possible to produce qualitative
differences in the direction of interstitial cell differentiation by supplementing
the culture media.
Several events were observed when a material isolated from Tubularia
hydranths was supplemented to the culture media. 'Late summer' animal
explants exhibited digestive cell attachment and migration. Interstitial
cell populations increased 2- to 3-fold. Interstitial cell derivatives (e.g. nerves,
cnidoblasts) were also more abundant in these cultures. Experiments are in
progress to quantitate these observations and to detect any changes in mitotic
activity occurring with the hydranth supplement.
M. G. wishes to express his sincere gratitude to Dr Allison L. Burnett for his stimulating
introduction to cnidarian development. This work was supported by a grant from Research
Corporation and by a USPHS Biomedical grant to Case Western Reserve University. A
portion of this work was completed while M.G. was a NSF Undergraduate Research
Fellowship recipient.
REFERENCES
M. (1965). Locomotory behavior of cells. In Cells and Tissues in Culture, vol. 1
(ed. E. N. Willmer), p. 194. New York: Academic Press.
ABERCROMBIE, M. (1967). Contact inhibition: The phenomenon and its biological implications. Natn. Cancer Inst. Monogr. 26, 249-277.
BROOKS, D., LESH-LAURIE, G. & GLICKMAN, M. (1971). Hydroid 'neurotrophic' material:
chemical and biological characterization. Am. Zool. 11, 347.
BURNETT, A. L. (1966). A model of growth and cell differentiation in Hydra. Am. Nat. 100,
165-190.
BURNETT, A. L. & HAUSMAN, R. E. (1969). The mesoglea of Hydra. II. Possible role in
morphogenesis. /. exp. Zool. 171, 15-24.
ABERCROMBIE,
Digestive cells in Tubularia
13
BURNETT, A., RUFFING, F., ZONGKER, J. & NECCO, A. (1968). Growth and differentiation of
Tubularia cells in chemically defined media. / . Embryol. exp. Morph. 20, 73-80.
CAMPBELL, R. (1967). Cell proliferation and morphological patterns in the hydroids Tubularia
and Hydractina. J. Embryol. exp. Morph. 17, 607-616.
COHEN, A. I. (1952). Studies on the pigmentation change during reconstitution in Tubularia.
Biol. Bull, mat: biol. Lab., Woods Hole 102, 91-99.
LESH, G. (1970). A role of inductive factors in interstitial cell differentiation in Hydra. J. exp.
Zool. 173, 371-382.
MARTIN, R. & TARDENT, P. (1963). Kultur von hydroides-Zellen in vitro. Rev. suisseZool.
70, 312-316.
PHILLIPS, j . H. (1961). Isolation and maintenance in tissue culture of coelenterate lines. In
Biology of Hydra (ed. H. M. Lenhoff & W. F. Loomis), pp. 245-251. Coral Gables: University of Miami Press.
SANYAL, S. &MOOKERJEE, S. (1960). Experimental dissociation of cells from Hydra. Wilhelm
Roux Arch EntwMech. Org. 152, 131-135.
TARDENT, P. (1962). Morphogenetic phenomena in the hydrocaulus of Tubularia. Pubbl.
Staz. Zool. Napoli 33, 50-63.
TARDENT, P. (1963). Regeneration in the hydrozoa. Biol. Rev. 38, 293-333.
TARDENT, P. & ERYMANN, H. (1959). Experimental^ Untersuchungen iiber den regenerationshemmenden Faktor von Tubularia. Wilhem Roux Arch. EntwMech. Org. 15, 11—37.
{Manuscript received 11 April 1972)