AMER. ZOOL., 14:773-781 (1974).

Regeneration in Medusa buds and Medusae of Hydrozoa

VOLKER SCHMID

Zoological Institute of the University of Zurich, 8006 Zurich, Switzerland

SYNOPSIS. Isolated medusa buds of Podocoryne carnea (Anthomedusa) can either de-
velop autonomously to functional medusae or transform to stolons and polyps. Up to
stage 8 the percentage of transformaiion can be idised 10 100% by icpeated Uaumaii-

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zation of the isolated buds. From stage 9 onwards, even traumatized buds always re-
generate medusae and never transform to polyps. At stage 8 the bud is differentiated,
lacking only the swelling of the outer mesoglea to become a normal medusa. A corre-
lation between the swelling of this mesoglea and the decrease of the percentage of
transformation to zero can be observed between stages 8 and 9. The presence of a
swollen mesoglea is not the only factor in stabilizing medusa structures, since, despite
removal of the mesoglea by the dissociation method, aggregates of dissociated buds of
stages 7 and 8 always formed medusa structures and never transformed to stolons or
polyps.
Regeneration of the manubrium in adult Hydromedusae shows a graded distribu-
tion, being highest in the center of the umbrella and decreasing rapidly towards the
circular canal. The extent of manubrium regeneration is not influenced by the amount
of wound material or the components of the gastrovascular s)stem.

Life cycles of most marine Hydrozoa
include a metagenic alternation of genera-
tions in which the sessile polyps represent
the asexually reproducing form, while the
sexual part of the cycle is limited to the
free swimming medusae. Amongst the colo-
nial polyps of the anthomedusa Podo-
coryne carnea (Athecata), which prefer-
entially settle onto snail shells, we find
besides feeding polyps (autostyles) the so-
called blastostyles which produce medusae
by budding. This process is based mainly
on local cell proliferation and immigra-
tion of somatic cells from the blastostyle
into the developing medusa bud (Brandle,
1971). The result of this developmental
process is a medusa which distinguishes
itself from the polyp from which it origi-
nates, not only by a different shape and
anatomical organization but also by a com-
pletely new set of somatic cells, not origi-
nally present in the polyp. They in-
clude amongst others cross-striated muscles,
medusae-typic nematocytes and particular
I thank Prof. Dr. R. Tardent for reading the
paper and his helpful criticism, and also the
"Schweiz. Nationalfonds zur Forderung der wis-
senschaftlichen Forschung" (Gesuch 3.205.69) for
supporting this work.
773
sensory cells. Each generation represents
a stable system as to structure and func-
tion, and both are the expression of the
same common genome, which we must as-
sume contains a portion controlling the
development of the polyp and another
governing that of the medusa. Hence, the
medusa development offers an excellent
opportunity for the study of differential
gene expression and of the factors respon-
sible for the maintenance and stability of
the differentiated state. One experimental
approach to these problems is the study of
the morphogenetic behavior in regenera-
tion. The literature concerned with regen-
eration in Hydrozoa and with the prob-
lems related to it deals mainly with the
polyps. Little is known about the recon-
stitutional abilities of medusae or their
developmental stages and how they com-
pare with the reconstitutional abilities of
the polyps. This paper is therefore mainly
concerned with the process of regeneration
in hydromedusae and their developmental
stages.
MATERIALS AND METHODS
The experiments were carried out on 2
marine colonial species: Podocoryne carnea
774 VOLKER SCHMID
(Anthomedusa, Athecata) and Campanu-
laria johnstoni* (Syn.: Phialidium haemi-
sphaericum, Leptomedusa, Thecata). The
colonies and medusae of Podocoryne
carnea were cultured in artificial sea
water (Aquarien-Grosshandel H. Wiegandt,
D-4150 Krefeld), whereas all investigations
on Campanularia were carried out at the
marine station of Villefranche-sur-mer,
France, on freshly collected medusae. The
latter were isolated from the zooplankton
and kept in large glass jars. They were
fed twice with Artemia salina before being
submitted to regeneration experiments.
Medusa buds of Podocoryne were iso-
lated from the blastostyle with ophthal-
mological scissors and, when necessary,
traumatized by squeezing them once or
three times at 2-hr intervals with forceps.
The same instruments were used for mak-
ing fragments of adult medusae of both
species.
Mechanical dissociation of medusa buds
and adult medusae was obtained by suck-
ing the animals repeatedly through a
pipette (for details, see Schmid, 1972).
For obtaining reaggregates the dissociated
cell masses were filtered and centrifuged
(2150g for 3 min). The reaggregates were
transferred for 4 hr into a drop of sea
water containing 0.01 mg/ml of strepto-
mycin. During this treatment the reaggre-
gation process was completed and the small
reaggregates were transferred to glass dishes
containing 20 to 30 ml of artificial sea
water.
For autoradiographic studies the speci-
mens were incubated in 3H-thymidine (1
/xCi/ml sea water) for 2 hr and after wash-
ing fixed in Carnoy's fixative (Schmid,
1972). The sections (5 yu.m) were covered
with Kodak NTB 3 emulsion, exposed at
4 C for 6 days, developed and stained with
Mayer's hemalum.
Specimens which were to be examined
with the electron microscope were fixed for
2 hr in 3i/2% glutaraldehyde and post-
fixed in 1% OsO4 for 1 hr. Both solutions
were made up with isotonic artificial sea
water (pH 8.1). After washing in sea water
[• Editors' note: Clytia johnstoni.]
the dehydrated specimens, embedded in
Araldite (Fluka AG, Buchs), were sec-
tioned with a Porter-Blum ultramicrotome.
The sections were mounted on copper
grids (75 mesh), stained with uranyl ace-
tate (alcoholic solution for 5 min) and
lead-citrate (15 min) and examined with
a Hitachi (HU-11 E) electron microscope.
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RESULTS
The morphogenelic behavior of trauma-
tized medusa buds of Podocoryne
carnea
The budding process of this species, ex-
tensively studied by Kiihn (1914) and
Weiler-Stolt (1960), was subdivided by
Frey (1968) into 10 developmental stages,
and these will be used in this paper.
Miiller (1913), Hauenschild (1956), Frey
(1968), and Schmid (1972) have studied
the developmental patterns of medusa buds
isolated from the blastostyle.
Such isolated buds either continue
autonomously their meduso-typic differen-
tiation or transform into stolons and
polyps. According to Frey (1968) and
Schmid (1972), medusa buds having
reached stage 3 or beyond are potentially
capable, when isolated, of continuing their
autonomous development, although most
young buds (stages 3-4) transform into
stolons or polyps. The rate of this regres-
sive transformation decreases with the in-
creasing age of the bud and falls to zero
once the development has reached stage 8.
From this critical stage onwards regres-
sive development is excluded.
When isolated buds between stages 1-8
of Podocoryne are repeatedly traumatized
by squeezing gently with forceps, the rate
of regressive transformation can be con-
siderably increased (Schmid, 1972). The
success of this measure is clone-specific and
can amount to 100% up to stage 7. It
declines slightly between stages 7 and 8
and falls to 0% between the stages 8 and
9. In some clones the rate of transforma-
tion remains 100% as far as stage 8. Stage
8, therefore, constitutes a clear-cut limit
beyond which even in traumatized buds
 REGENERATION IN MEDUSA BUDS AND MEDUSAE 775

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FIG. 1. a, Presumptive striated muscle cell region of stage 8; 10.400X. G = golgi apparatus; iM =
of a medusa bud o£ stage 3; 15,600x. b, Striated inner mesoglea; M = mitochondria; Mf = niyofila-
muscle cells of the subumbrella of a medusa bud ments; N = nucleus.
776 VOLKER SCHMID
development will always proceed in a
meduso-typic way. Therefore, stage 3 and
stage 8 seem to be important thresholds
in the course of medusa development.
Structurally, these stages can be charac-
terized as follows.
Stage 3 constitutes a primordial stage
in which the anlage of the manubrium
and the radial canals are visible, but in
which primordia of tentacles, circular
canals, velum or subumbrella are anatomi-
cally not yet defined. The bud primordium
of this stage, when examined with the
electron microscope, is composed of cells
which have an embryonic aspect. In the
presumptive subumbrellar area, no myo-
T ® •W^-'":-' i'^'-V-'-^'r?M
S
1SJP
Ri
FIG. 2. Arcdian section through a medusa bud of
stage 8" 150X. Ex = exumbrella; G = gonads;
M = muscles; Ma = manubrium; N = nematocytc
filaments can be observed (Fig. la).
In stage 8, in which cell proliferation
within the bud has come to a standstill,
differentiation of meduso-typic cells is
ultrastructurally completed. The subum-
brella contains cross-striated muscles (Fig.
\b) and all other parts of the medusa are
visible (Fig. 2). The assumption that the
differentiation is completed is supported
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by the fact that the bud development,
when exposed to actinomycin D, can be
inhibited only up to stage 7. The older
stages, which are refractory to the action
of actinomycin D, are characterized by a
rapid swelling of the mesoglea. Once this
has occurred the medusa is functional and
•fiS&^k^:<M
Ma
^':VO!- ^l;"W'i
G
Ra
ring; Ra = radial canal; Ri = circular canal; S:-
subumbrellar plate; T = tentacles; V - velum.
(After Schmid, 1972.)
 REGENERATION IN MEDUSA BUDS AND MEDUSAE
STAGE 9
FIG. 3. The relation of the transformation rate of
isolated traumatized buds to the thickness of the
swelling mesoglea. Ordinate, percentage of trans-
formation; abscissa, thickness of the mesoglea in
microns. (Redrawn after Schmid, 1972.)
detaches from the blastostyle. There seems
to be a causal relationship between the
swelling of the mesoglea on the one hand
and the loss of the faculty for regressive
transformation of the bud on the other
(Fig. 3). The mesoglea once formed ap-
pears to stabilize the differentiated medusa
cells in a meduso-typic manner. The sta-
bility of the differentiated state can no
longer be disturbed from stage 8 onwards,
FIG. 4. Differentiations of aggregates prepared from
medusa buds (a) and adult medusae (b), 60X-
E = eggs; MA = manubrium; Ne = nematocytes;
777
even if the isolated bud is repeatedly trau-
matized. The following experiments have
been designed mainly to examine the pos-
sible role of the mesoglea as a stabilizing
factor.
The behavior of dissociated medusae
and medusa buds
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Medusae and their developmental stages
of Podocoryne were dissociated mechani-
cally. The morphogenetic behavior of the
reaggregated cell masses is shown in Fig-
ure 4. These reaggregates generally express
themselves morphogenetically by develop-
ing either medusa-like or stolonial struc-
tures. The latter case, which represents a
regressive transformation, is less frequent
than the reconstitution of medusae struc-
tures. The rate of regressive transformation
is not higher than in intact, non-trauma-
tized isolated medusa buds (see above).
In 6 out of 4900 cases, however, aggregates
obtained from dissociated adult medusae
produced stolons.
These results clearly show that regres-
sive transformation from medusae struc-
tures to stolonial formation cannot be
enhanced by dissociation and reaggrega-
tion, which completely remove the meso-
Pa = periderm of the aggregate; P = periderm of
the stolon; Pk = polyp bud; St = stolon; TA =
tentacles. (After Schmid, 1972)
778 VOLKER SCHMID
glea. The mesoglea, therefore, cannot play
a predominant role in stabilizing the
medusa cells in their state of differentia-
tion. We must, therefore, look for other
stabilizing factors.
Regeneration in adult medusae
According to the literature, the recon-
stitutional abilities of Hydromedusae (es-
pecially of Anthomedusae) are very much
restricted as compared to those of the
asexual polyps.
Recent investigations on the regeneration
potential of Antho- and Leptomedusae
(Schmid and Tardent, 1971) revealed an
astonishing ability of these animals to re-
generate missing parts and organs. The
experiments reported here intend to show
that the regenerative abilities are unevenly
distributed throughout the animal.
FIG. 5. Schematical drawing of the fragmentation
technique for hydromedusae. a, Removal of the
manubrium with varying amounts of the umbrella
tissues, b, Manufacture of interradial fragments of
Campanularia johnstoni. x,, x2, x3 = lines of sec-
tion; a, b, c, d = fragments of umbrella; cc =
circular canal; G = gonad; M = manubrium; re =
radial canal.
In this comparative study we used the
Anthomedusa Podocoryne and the Lepto-
medusa Campanularia. The anatomical
and cellular architecture of both medusae
is in many respects quite different. These
differences mainly concern the subumbrel-
lar tissues, the gonads and the sensory
organs.
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In a first series of experiments, per-
formed on both Podocoryne carnea and
Campanularia johnstoni, the central part
of the umbrella including the manubrium
and an adjacent portion of the umbrella
of varying size was extirpated by a circu-
lar cut. These central isolates were dis-
carded, while the morphogenetic behavior
of the ringlike, marginal portion of the
umbrella was examined. In the case of
Podocoryne these discarded isolates lacking
the peripheral part represented 14, i/>, and
% of the bell. In Campanularia the cor-
responding pieces amounted to 1/2 and 3/4
of the bell (see Fig. 5a).
In all cases the central hole is quickly
closed, reconstituting the bell- or disk-like
shape of the isolate. This wound-closure
is followed by the regeneration of the miss-
ing manubrium in the center of the bell.
In Campanularia this regenerated manu-
brium is ready to feed 2 to 3 days after
the operation, while in Podocoryne the
manubrium becomes functional, but not
before 5 to 7 days after the operation.
In addition Podocoryne, when fed, will
also regenerate the gonads on the manu-
bria.
Table 1 indicates that the rate of manu-
brial regeneration on marginal umbrellar
fragments is higher the larger the isolates
are. In order to examine whether the in-
crease in the rate of regeneration depends
upon the amount of gastrovascular com-
ponents (radial- and ring-canals) present
or upon some other factors, we isolated
in Campanularia interradial sectors of the
umbrella. As shown in Figure 5b, these
fragments were cut out in a way to vary
the length of the total cut surface, the
amount of umbrellar organs like circular
canals, or the regional quality of the sub-
umbrellar tissues including the acellular
 REGENERATION IN MEDUSA BUDS AND MEDUSAE 77!)

TABLE 1. The rate of manubrium regeneration in Hydromedusae m relation to the amount of

removed umbrella.

Umbrella parts Podocoryne Campanularia

removed with carnea jonnstoni
the manubrium n % formed manubria n /o formed manubria
<rr

V4 45 75.5
14 75 46.5 10 100
59 15.3 10 0

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mesoglea. In order to exclude individual
or colonial variability the four fragments
a-d were always isolated from the same
animal. The four different types of frag-
ments quickly restored the circular shape
of a medusa (Schmid and Tardent, 1971).
This process was accomplished 6 to 8 hr
after isolation. Most fragments (a-c) re-
generated a functional manubrium 3 to 4
days after isolation. The results of these
experiments, given in Table 2, permit
the following conclusions.
An increase of the cut surface or the
reduction of the circular canal or the total
absence of radial canals does not signifi-
cantly influence the rate of manubrial
regeneration.
The fragments c and d (Fig. 5b) were
made as equal as possible with the excep-
tion of the amount of mesoglea present.
Fragment c contains the thick central por-
tion of the mesoglea, while fragment d
includes the thinner peripheral part of
the mesoglea. It is in these fragments con-
taining the smaller amount of mesoglea
where the rate of manubrial regeneration
is at its lowest.
These results clearly demonstrate that
neither the length of cut surface nor the
amount of circular or radial canals present
TABLE 2. The rate of manubrium regeneration in
interradial fragments of different shape in
Campanularia johnstoni.
% of regenerated
Fragment manubria
31 73
31 65
31 74
31 29
a, b, c, d = different fragments; for detail, see
Figure 5b.
in the fragment can be made responsible
for the decrease of the regenerative poten-
tial from the center of the umbrella to-
wards its margin.
DISCUSSION
The regenerative behavior of the dif-
ferent stages (larvae, stolons, polyps, me-
dusae) in the metagenic cycle is not at
all the same, although the genome is the
same throughout. The expression of the
genome in each stage appears, therefore,
to be very stable in the reconstitutional
processes, especially when polyps and me-
dusae are closely considered. There have
never been reports of a polyp regenerating
a medusa instead of a missing hypostome
or vice-versa. A system of stabilizing ele-
ments seems to ensure the differentiation
of embryonic cells (interstitial or undiffer-
entiated cells) in the appropriate direc-
tion. These stabilizing elements could be
mainly cell-inherent, or based on cell-cell
or tissue-tissue interactions. This paper
first has given an indication of when in
the development of the medusa bud a
change in stability is expressed in the re-
generative behavior, and second, presents
some results concerning regeneration in
medusae.
In the budding process an organism de-
velops with a completely different struc-
tural organization from the polyp which
forms the bud. The process of budding is
strongly proliferative (Weiler-Stolt, 1960) ;
however, an immigration of cells was also
reported (Brandle, 1971). According to
the isolation experiments of Frey (1968)
and Schmid (1972), very young buds of
stage 3 with only rudimentary anlagen of
780 VOLKER SCHMID
some medusa organs are able to develop
autonomously. It seems possible, therefore,
that the bud is completely self organizing
almost from the very beginning of its
development but remains dependent on the
polyp for nutrition. This power of self
organization is excellently demonstrated
in aggregates of stages 3 and 4-5 which
started without striated muscles or ten-
tacles but were able to build up these
structures after some days (Schmid, 1972).
The majority of isolated buds of young
stages regenerate stolons which can form
polyps after a process called transforma-
tion. This ability of isolated buds to trans-
form into polyps decreases, the older and
more differentiated the isolated buds are,
and ceases between stages 8 and 9. Al-
though the portion of buds undergoing
transformation can be significantly in-
creased in early stages by repeated trau-
matization, it has never been possible to
pass the line between stages 8 and 9. This
method, therefore, perhaps indicates that
the system responsible for the stabilization
of medusa structures is activated between
these two stages. A detailed study of stage
8 revealed that the bud is fully differen-
tiated, awaiting only the swelling of the
outer mesoglea to be a functional medusa.
Indeed a strong correlation exists between
the swelling process of the mesoglea and
the pronounced decrease in the percentage
o£ transformation. A possible role for the
mesoglea in the stabilizing system of the
medusae has to be considered since, in
every case in which medusae have been
transformed into stolons and polyps
(whether induced by traumatization or
other methods), the mesoglea has always
been removed (Schmid, 1972). It is pos-
sible that collagens, of which the mesoglea
is composed, are involved in morphogenetic
processes as is sufficiently documented from
vertebrates (Wessells and Cohen, 1968).
The dissociation experiments, however,
prove that although the mesoglea was no
longer present a transformation to polyps
practically never occurred except in the
case of aggregates from very young buds.
Thus, there must be other factors beside
the mesoglea in the stabilizing system and
it seems that the derivatives of the glocken-
kern are also engaged in a similar function
(Schmid, 1972).
As soon as the bud has passed the line
between stages 8 and 9, it will always
regenerate medusa structures after trau-
matization. The regenerative power of the
completed medusa is enormous. Fragments
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of the umbrella of Campanularia lacking
any components of the gastrovascular sys-
tem (and therefore free of interstitial cells)
can essentially reconstitute a whole medusa
(Schmid and Tardent, 1971). From stud-
ies of polyps, especially Hydra, consider-
able knowledge has been gained about the
factors which influence the regenerative
events. In the case of medusae little is
known. Gradients and polarity may play
a role, but when or to what extent is un-
certain. The results in the second part of
the paper, although only preliminary,
clearly demonstrate that in medusae a
gradient in manubrium regeneration exists.
This gradient decreases from the center
towards the periphery of the animal.
Neither the amount of wound surface nor
the portion of circular canal (and with it
the quantity of interstitial cells) seems to
influence the event of manubrium regen-
eration. Therefore, it appears that the
number of interstitial cells is of minor
importance in this regenerative process
and a strongly favored possibility, sup-
ported by evidence from studies of polyps
(Burnett et al., 1966), is that the regenera-
tion of the manubrium proceeds by a
process of metaplasia. In speculating about
the nature of the factors which are respon-
sible for the pronounced difference in the
ability to regenerate manubria (fragments
c and d), a gradient of substances or a
difference in electric properties (Rose,
1970) of the subumbrellar tissues must
be considered. Indeed, it is felt that the
structure and composition of the mesoglea,
which are the only visible differences be-
tween fragments c and d, also deserve more
penetrating investigations.
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