403
Journal of Cell Science 108, 403-412 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
Novel tools for the study of development, migration and turnover of
nematocytes (cnidarian stinging cells)
Jakob Weber
Zoological Institute, University of Zurich-Irchel, Winterthurerstr. 190, CH-8057 Zurich, Switzerland
SUMMARY
The rhodamine derivatives tetramethyl-rhodamine-5/6maleimide (TROMI) and tetramethyl-rhodamine-6-isothiocyanate (TRITC) were allowed to react with living
Hydra vulgaris. The two fluorescent dyes stain the polyps
to different degrees, apparently without impairing their
viability and behaviour. Concerning nematocytes, TROMI
preferentially couples to cytoskeletal elements only of
mounted nematocytes whereas TRITC selectively reacts
with structural components of cysts of late nematoblasts,
which thereafter develop apparently normally into mature
nematocytes. Hence TROMI-labelling indicates that nematocytes are mounted and ready for discharge; TRITC-
labelling can be used as a tool to investigate the final maturation, migration and installation of nematocytes in
Hydra.
Together with a new non-fixative method to dissociate
Hydra polyps into single, identifiable cells, the two labelling
methods allow direct quantitative dynamic studies of
nematocyte turnover and open new possibilities of investigating the regulation and the mechanisms of nematocyte
supply and migration.
INTRODUCTION
scattered along the whole animal and are most abundant in
the tentacles. The formation of nematocytes begins with the
proliferation of committed stem cells (interstitial cells)
located in the ectoderm of the body column to nematoblasts; it continues with the genesis and assembly of the
complex intracellular cyst, the migration to and installation
at their ultimate destination and, finally, ends with the
discharge of the cysts into prey or aggressors, or displacement by new cells (for reviews see Campbell, 1988a,b; Bode,
1988).
Studies on the overall temporal succession of the events in
the nematocytes’ life (David and Gierer, 1974), morphological
studies on the early development of nematoblasts (Slautterback
and Fawcett, 1959; Holstein, 1981) and descriptive reports on
nematocyte migration (Günzl, 1971; Campbell and Marcum,
1980), and distribution and turnover (Zumstein, 1973;
Herlands and Bode, 1974; Bode and Flick, 1976; Weber et al.,
1978), have been published. However, the methods used in
these studies, namely, the direct observation of nematocysts in
intact tissue, the radiolabelling of nematocytes and the maceration of tissue, which abolishes subcellular fine structures, limit
the extent of such investigations considerably. In addition,
some studies took advantage of grafting with epithelial (i.e.
interstitial cell-free) Hydra, which, though elegant and illustrative, does not reflect the real situation. Therefore detailed
insights into developmental or regulatory aspects (see Bosch
and David, 1991), and mechanisms involved in migration (see
González Agosti and Stidwill, 1991, 1992), as well as into the
underlying principles of the regulation of genesis and life of
all four types of nematocytes occurring in Hydra, are mostly
Nematocytes, the stinging cells of Cnidaria (polyps, jellyfish,
corals), are often characterised as the most elaborate and
complex cells found in multicellular organisms. They occur in
large numbers and many variants in all species of the Phylum
Cnidaria and serve mainly for food capture and defence against
aggressors (for reviews see Blanquet, 1977; Mariscal, 1974,
1984; Burnett and Calton, 1977, 1987; Tardent et al., 1985;
Hessinger and Lenhoff, 1988; Williams et al., 1991).
For several reasons nematocytes and their progenitors, the
nematoblasts, are excellent objects, not only for studying toxinological, mechanistic and signal transductional aspects of the
exocytotic discharge of nematocysts but also for investigating
more general cell biological phenomena of multicellular
organisms, particularly of the control and the mechanisms of
cell differentiation, of cell migration or the regulation of cell
numbers at the level of the live and intact organism: the differentiation pathway of nematocytes is well defined and not very
long, nematocytes are relatively easy to observe and manipulate in vivo and can, due to their prominent intracellular
capsule (the nematocyst), be identified rather easily amongst
other cells. The organisms in which nematocytes occur are relatively primitive and therefore in some respects easier to study
than, e.g. the more highly evolved insects or vertebrates. In
addition, some freshwater and marine cnidarians can easily be
kept in large numbers under laboratory conditions.
The freshwater polyp Hydra vulgaris (Campbell, 1989)
consists of a closed diploblastic body column with a gastric
cavity and a crown of tentacles. Nematocytes are found
Key words: Hydra, nematocyte, nematocyst, development,
migration, covalent labelling, rhodamine
404
J. Weber
lacking. Even less is known about nematocytes from other representatives of the Cnidaria.
In this paper methods for the selective in vivo fluorescentlabelling of developing and of mounted nematocytes are
presented. Furthermore, a new and convenient method to dissociate Hydra polyps under non-fixative conditions into single
cells is introduced. These methods allow the direct study of the
development of later stages of nematoblasts and the turnover
of nematocytes.
MATERIALS AND METHODS
Materials
Cultures of Hydra vulgaris (Hydrozoa; Cnidaria) (Campbell, 1989)
were grown in culture solution as described (Loomis and Lenhoff,
1956; Weber et al., 1987) and fed four times per week with Artemia
larvae. For all experiments and analyses randomly chosen specimens
from these mass cultures were used.
All reagents used were of analytical grade.
Dissociation of Hydra polyps into single cells
A small volume of culture solution containing 3 to 10 Hydra polyps
at 14°C was pipetted into 1 ml of dissociation medium (see below),
preheated to 40°C. After reducing the total volume to approx. 60 to
200 µl, the polyps were incubated for 10 minutes at 40°C and subsequently dissociated mechanically to form a suspension of single cells
(or cell clusters) by vigorously shaking the tubes. The degree of dissociation was controlled visually; too-intense shaking resulted in an
increase in cell fragments, too-gentle shaking yielded larger tissue
pieces. The dissociated cells were held on ice and examined under a
Reichert-Jung Polyvar microscope equipped with Nomarski differential interference (DIC) optics. Pictures of individual cells as presented
in Fig. 1 were recorded with a video camera mounted on a Polyvar
microscope and transferred on-line to a Macintosh computer. The
pictures were processed and arranged with the software program
Adobe Photoshop 2.5.1.
The composition of the dissociation buffer, which was routinely
used for identification and/or counting of different stages of nematoblasts and nematocytes, was 137 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, 1.8 mM KH2PO4, pH 7.2 (PBS). In some experiments a
dissociation medium, made according to Gierer et al. (1972), and supplemented with 54 mM KCl, was used to improve the (osmotic)
stability of the epithelial cells. If large polyps have to be dissociated
it is sometimes advisable to cut the animals into smaller pieces prior
to the 40°C incubation, since the endodermal cells are held tightly
together by the massive acellular mesoglea.
In vivo labelling of Hydra with TRITC or TROMI
Hydra polyps were incubated in Hydra culture solution including 1
µM tetramethyl-rhodamine-6-isothiocyanate (TRITC; Molecular
Probes) or 25 µM tetramethyl-rhodamine-5/6-maleimide (TROMI;
Molecular Probes) for 30 minutes at room temperature. These concentrations proved to be optimal for our purposes and apparently did
not affect the polyps’ health (see Results). After washing the polyps
by repeatedly transferring them into new, dye-free culture solution,
they were kept and fed as described for untreated animals.
Stock solutions (1 mM) of TRITC and TROMI in water were stored
frozen at −20°C up to several weeks without an observable reduction
in the chemical reactivity. They were diluted with Hydra culture
solution immediately before use.
Analysis of nematoblasts and nematocytes in labelled
Hydra
Isolated nematocysts
After different periods of time after TRITC-labelling about 20 intact
Hydra (or polyps separated into tentacles including hypostome and
body column excluding buds) were frozen at −20°C in a small volume
of water. Cell-free nematocysts were isolated from these frozen
probes as previously described (Weber et al., 1987; Weber, 1990)
using 70% Percoll (Pharmacia). The purified nematocysts were
pipetted onto glass slides, dehydrated in a vacuum desiccator and
stored in a dry state.
The amounts of labelled and unlabelled nematocysts of the different
types were determined after mounting the isolated cysts in 40%
poly(ethylene glycol) 1550 (PEG; Serva), 5% propyl gallate (Fluka;
Giloh and Sedat, 1982), 2 mM Tris-HCl, pH 9, under a Reichert-Jung
Polyvar microscope equipped with Nomarski-DIC and epifluorescence illumination (a filter set designed for rhodamine fluorescence
was used). Addition of PEG to the mounting medium permits discrimination between fully mature and immature nematocysts, since
cysts that are not yet under the full internal osmotic pressure shrink
anisotropically and display irregularities in their shape (see Weber,
1989). Totals of 300 to 500 undischarged and mature nematocysts of
each type (stenoteles, desmonemes, isorhizas) were counted and classified as labelled or not. The criterion for a cyst as being labelled was
in all three cases a clearly identifiable fluorescent internal tubule (see
Results).
Single nematocytes
TRITC- or TROMI-labelled Hydra were dissociated into single cells
as described above. After removing most of the epithelial cells by a
2 minute centrifugation at 2,000 g in 50% Percoll in dissociation
buffer (0°C), the remaining, sedimented cells (mostly interstitial cells,
nematoblasts, nematocytes, some gland cells) were immediately
analysed as a cell suspension in dissociation buffer and classified as
outlined above for isolated nematocysts (100 to 200 stenoteles and
isorhizas were routinely counted). Since some late-stage immature
nematocytes are difficult to distinguish optically from mature nematocytes, the counts may include some immature nematocytes (see
Results). TROMI-labelled cells were identified by their highly fluorescent bundles of rods (see Results). In some experiments tentacles
and body columns were analysed separately.
Intact tissue
Qualitative studies with TRITC- or TROMI-labelled Hydra were
performed after fixation for 10 minutes in absolute ethanol. The fixed
polyps were gradually rehydrated, their pedal disks and tentacle rings
were dissected, the body column was opened with fine tungsten
needles and the endoderm stripped off the body column and discarded.
Tentacles and body column ectoderm were mounted in PEG-propyl
gallate-Tris (see a above) and examined in the fluorescence microscope. The fixed animals were studied within 2 hours following
fixation, since the distribution of the fluorescence changes slightly
within hours after fixation.
Ethanol fixation proved to be superior to other fixation methods,
since the discharge of nematocysts is minimal.
Transplantation of Hydra polyps
At 0, 24 or 48 hours after TRITC labelling, the distal (upper) halves
of unlabelled polyps were grafted onto the proximal (lower) halves of
labelled polyps (Tardent, 1966). Only bud-less polyps were used.
After 24 or 48 hours of parabiosis, the polyps were fixed for 10
minutes in absolute ethanol, gradually rehydrated and then mounted
in PEG-propyl gallate-Tris and examined microscopically for fluorescent nematocytes that had migrated into the unlabelled tissue.
RESULTS
Dissociation of Hydra into single cells
Hydra polyps can be macerated (David, 1973) and the various
types of cells can be easily identified, classified, counted and
Novel tools for studying nematocytes
autoradiographed. However, intracellular organelles and fine
structures are disrupted; furthermore, different stages of nematocyte development are difficult to identify and mature nematocysts may lose some of their content (Weber, 1994). On the
other hand, if polyps are dissociated (Gierer et al., 1972), the
viable single cells obtained are very fragile and not suited for
quantitative studies or fractionation (see Greber et al., 1992).
Therefore, the development of an alternative, gentle method
for dissociating polyps rapidly and quantitatively into single
cells was desirable. Incubation of Hydra in salt solutions of
moderate ionic strengths (see Materials and Methods) at
elevated temperatures (routinely 40°C) for a few minutes
permits the dissociation of polyps (including their tentacles)
into single cells and/or, depending on the intensity of the
mechanical disruption, into groups of tightly associated cells
such as battery cells or clusters of nematoblasts (Fig. 1). The
different cell types of Hydra (Bode et al., 1973; David, 1973)
are easily identifiable and observed at amounts equivalent to
those obtained by maceration; only nerve and sensory cells
were more difficult to identify, since after rapid (partial) retraction of their processes they become, in many cases, indistinguishable from interstitial stem cells. Fig. 1 shows a collection
of abundant cell types after the dissociation of whole polyps;
nematocytes and different developmental stages of nematoblasts can readily be identified and quantified.
Under the conditions used, the dissociated cells are subvital;
405
i.e. despite frequently observed vigorous active shape changes,
they cannot reaggregate and regenerate new polyps (Gierer et
al., 1972; Flick and Bode, 1983). Within the first hours after
dissociation, however, their intracellular topological organisation and physiology seem not to be grossly affected. By raising
the temperature during dissociation to 50°C, cells are obtained
in more ‘fixed’ state, and by using salt solutions of different
compositions, the (osmotic) stability of particular cell types
can be optimised. Cells are kept for at least 2 hours in a good
state and were, for example, fractionated within this period by
density gradient centrifugation in Percoll-containing dissociation buffer.
By this procedure tentacles are dissociated into single endodermal epithelial, ectodermal battery (ba in Fig. 1) and
nerve/sensory/interstitial cells. After more vigorous dissociation, nematocytes (is, st and de in Fig. 1) are gradually lost
from the battery cells and under the conditions used here, most
of the stenoteles (approx. >80 to 90%) are no longer found
within the battery cells.
Labelling of living Hydra by rhodamine B derivatives
The two rhodamine derivatives tetramethyl-rhodamine-isothiocyanate (TRITC) and tetramethyl-rhodamine-maleimide
(TROMI) were chosen for covalent labelling of Hydra (see
also Klimek, 1979).
TRITC (1 µM) added to Hydra culture solution, diffuses
Fig. 1. Collage of dissociated cells of whole Hydra. Polyps were dissociated and the single cells were recorded as described in Materials and
Methods. The selected cells shown are identified as ectodermal (ec) and endodermal (en) epithelial cells, gland cell (gl), nerve cell (ne),
tentacular battery cell (ba), pair of i-cells (pi; presumably stem cells), and different stages of nematoblasts (nb1: 16- or 32-cell cluster of very
early nematoblasts; nb2: part of a more developed cluster, presumably of desmonemes; nb3 and nb4: parts of clusters of later stages of stenotele
nematoblasts) and the nematocytes, stenotele (st), desmoneme (de), holotrichous isorhiza (is).
406
J. Weber
readily across cell membranes and couples at varying degrees
to most of the tissue components of both the ecto- and the
endoderm of polyps. There is no evidence that the animals are
affected negatively in their behaviour or morphology by the
procedure; even several months after labelling the polyps still
fluoresce to a considerable degree. In certain populations of
nematoblasts the growing cysts are strongly and selectively
labelled by TRITC: late-stage nematoblasts (see below)
display highly fluorescent walls and shafts, tubules and
adhering structures, whereas in earlier nematoblasts as well as
in mature nematocytes only a negligible amount of the reactive
dye couples to the cyst’s wall. TRITC-labelled nematoblasts
develop and mature normally and apparently without delay into
fully functional nematocytes.
TROMI (25 µM) in contrast to 1 µM TRITC, labels living
Hydra only weakly, and most of the incorporated fluorescence
Fig. 2. Labelling of Hydra with TROMI.
Hydra were labelled with TROMI and
subsequently fixed and mounted as
described in Materials and Methods. The
plane of focus for the fluorescence
pictures (upper row) lies close to the
apical part of the cell surface of the
nematocytes, and the focus of the
corresponding Nomarski-DIC pictures
(lower row) was adjusted to deeper
within the tissue in order to be able to
identify the types of nematocysts.
(A) Four battery cells in a tentacle (with
numerous desmonemes, some isorhizas
and 3 of them with a prominent stenotele
as indicated by the arrowheads); and (B)
parts of the body surface (with some
holotrichous isorhizas that are mounted
perpendicular to the plane of focus and 2
stenoteles, which are marked by
arrowheads). Bar, 20 µm.
is detected associated with presumably endosomal compartments of ectodermal epithelial cells. As with TRITC, the
animals survive without behavioural or morphological
anomalies. The most striking feature with TROMI labelling is
its selective reaction with cytoskeletal elements of mature
nematocytes that are in direct contact with the outer milieu, i.e.
with those nematocytes that are mounted.
If the labelling concentration of TRITC is raised to 5 µM
and higher, the staining becomes more intense than with 1 µM
dye, but the polyps show temporary degenerative effects;
TROMI seems to affect the polyp’s health only at concentrations exceeding 100 µM.
If ethanol-fixed and thus permeabilised Hydra are reacted
with TROMI, a pronounced and general increase in the fluorescence of the tissue, as compared to the in vivo labelling, is
observed. In contrast, TRITC labelling in living and fixed
Novel tools for studying nematocytes
polyps is quantitatively and qualitatively similar. The different
staining properties of TRITC and TROMI, therefore, seem to
be based on the different potencies of these reagents to cross
cellular membranes and not on their different intrinsic
chemical reactivities (TRITC preferentially reacts with amines,
TROMI with sulfhydryls). The pronounced selectivity of
TRITC for later nematoblast stages, in contrast, obviously
reflects a higher chemical reactivity of the cysts of these cells
as compared to the cysts of earlier nematoblasts or mature
nematocytes.
Labelling with TROMI
Fig. 2 shows parts of a tentacle (A) and of a body column (B)
of TROMI-labelled Hydra polyps under fluorescence and
Nomarski-DIC illumination. In the tentacle as well as in the
body column, those nematocytes that are on the surface of the
tissue (and are thus presumably mounted) show highly fluorescent cnidocils (sensory cilia) and intracellular basket-like
structures. The fluorescence labelling pattern is retained after
dissociation of the polyp into single cells (not shown) and is
stable for at least seven days. The labelled intracellular structures correspond to the so called rods, uncharacterised integral
components of the tubulin and actin polymer-containing
complex basket that envelopes the nematocysts (Stidwill and
Honegger, 1989). The functionality of the nematocytes seems
not to be affected by the labelling.
During the course of this work, immediately after TROMI
labelling, neither fluorescent nematocytes apart from the
surface of the polyp nor non-fluorescent nematocytes that were
obviously mounted were found.
The relative amount of labelled nematocytes in the body
columns of Hydra was determined by dissociating polyps
immediately after the TROMI incubation and counting the
amount of fluorescent cells (Table 1). Labelled and thus
mounted desmonemes or atrichous isorhizas were observed
rarely, if at all, in agreement with published work (Bode and
Flick, 1976). The amounts of labelled stenoteles and holotrichous isorhizas were determined as 26±6% and 85±6%,
respectively, in the body columns (Table 1). Both values
fluctuate considerably even within the same clone of Hydra
(see inset of Fig. 3, and the broad ranges in Table 1), which
indicates that the number of mounted nematocytes can vary
extensively (see Zumstein, 1973; Bode and Flick, 1976).
The amount of labelled nematocytes in the body column at
various times after a single incubation with TROMI is shown
in Fig. 3. For stenoteles and holotrichous isorhizas the labelling
frequency initially decreases by about 20 to 30% per day and
later about 10% per day compared with freshly labelled
controls. These results are consistent with the assumption that
TROMI labels only mounted nematocytes, which are then
gradually lost within one to two weeks (Bode and Flick, 1976).
Labelling with TRITC
Fig. 4A to C shows different parts of the body column
ectoderm of freshly TRITC-labelled Hydra by fluorescence
and Nomarski-DIC illumination. Although most tissue components are fluorescent, nematoblasts in which the growing
capsule itself is heavily labelled are clearly observable. Fig. 4A
shows three fluorescent stenotele nematoblasts (triangles) out
of a larger cluster, Fig. 4B various clusters of fluorescent
desmoneme nematoblasts, and Fig. 4C two single, nearly
mature, fluorescent stenotele nematoblasts (triangles). Mature
nematocytes (see the single stenotele marked with an asterisk
in 4A) and earlier stages of nematoblasts (see the two stenotele
nematoblasts with asterisks in 4C), however, are strikingly
non-fluorescent. A careful inspection of body columns and of
dissociated single cells of labelled polyps confirms that neither
very early stages of nematoblast nests nor fully mature nematocytes of any type display the typical brightly fluorescent
pattern of later nematoblasts shown in Fig. 4A-C. In particular, no TROMI-positive and/or no tentacular nematocytes were
found to be fluorescent immediately after the TRITC treatment.
The ability to be labelled by TRITC seems to be restricted to
clusters of nematoblasts of which the future capsule type is
clearly identifiable and whose capsular wall has lost its pronounced fragility. This labelling ability is lost shortly after the
disintegration of the nematoblast nests.
Fig. 4D shows cell-free nematocysts isolated five days after
labelling of living polyps with TRITC. Two stenoteles, two
Nematocytes
Stenoteles
Desmonemes
Holotrichous isorhizas
Atrichous isorhizas
Mean (%)
s.d. (%)
Range (%)
Counts
26
<5*
85
<5*
6
16-38
23
6
75-94
16
Hydra were reacted with TROMI and the body columns of the polyps were
dissociated as outlined in Materials and Methods, and examined with a
fluorescence microscope. For each count 100 to 200 nematocytes of the
indicated type were classified as labelled or not labelled. The data were
collected over the range of two months.
*Found only rarely and erratically; possibly cross-contaminations from
tentacular tissue.
AAAAAAAAAA
AAAAAAAAAAAAAAAAAA
AAAAAAAAAA
AAAAAAAAAAAAAAAAAA
AAAAAAAAAA
AAAAAAAAAAAAAAAAAA
AAAAAAAAAA
AAAAAAAAAAAAAAAAAA
AAAAAAAAAA
AAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAA
AAA
AAAAAAAAAAAAAAAAAA
AAA
AAAAAAAAAAAAAAAAAA
AAA
Relative amount of labelled
100
nematocytes in the body column (%)
80
100
60
40
80
60
40
20
0
0
2
4
6
Stenoteles
holotr. Isorhizas
20
Table 1. Relative amount of TROMI-labelled nematocytes
in the body column of Hydra
407
F
F
F
F
0
0
1
2
3
4
5
Time after TROMI-labelling (days)
6
7
Fig. 3. Decrease in fluorescent nematocytes within several days after
TROMI labelling. At 0, 1, 2, 5 and 7 days following incubation of
Hydra with TROMI (see Materials and Methods), the body columns
of 20 polyps were separated. Ten of them were used as controls and
were additionally labelled with TROMI (filled symbols in the inset),
the others were used directly (open symbols in the inset). After
dissociation into single cells, the fractions of labelled stenoteles
(squares) and holotrichous isorhizas (circles) were determined and
plotted in the inset. The main graph was obtained by referring the
obtained values to the respective controls, which were set to 100%.
Hydra were fed where indicated (F).
408
J. Weber
desmonemes and one isorhiza in this figure display highly fluorescent intracapsular structural components whereas in one
stenotele only the surrounding wall is slightly fluorescent. The
former have, without losing their fluorescence, obviously
developed from labelled nematoblasts, whereas the latter was
already mature at the time of the TRITC labelling (see also Fig.
5).
In isolated mature nematocysts (Fig. 4D) the TRITC fluorescence is not abolished after incubation of the cysts in 1 M
sodium hydroxylamine-hydrochloride, pH 8, or 0.5 M sodium
glycinemethylester-hydrochloride, pH 10, indicating that the
dye is not coupled via sulphydryl groups (as could be assumed
from their high content in walls and tubules (Blanquet and
Lenhoff, 1966; Kurz et al., 1991) but rather via amino groups
(Drobnica et al., 1977)).
The TRITC reaction is not a strict pulse-labelling, since
hours to days after washing out the dye nematoblasts entering
the critical, TRITC-susceptible stages become labelled to some
degree, presumably by unreacted dye stored within the Hydra
tissue. Furthermore, the fluorescence intensities of all labelled
cysts become somewhat reduced during the final maturation
phase by either loss or chemical derivatisation of the dye or by
quenching (compare the fluorescence intensities of the two
nearly mature stenoteles in Fig. 4C with the three earlier stages
in Fig. 4A, which are all marked with triangles).
Fig. 5 shows the time course of the appearance of mature
fluorescent nematocysts at various periods of time after TRITC
labelling of living Hydra. The amount of cysts that are fluorescent initially lies between 0 and 10% for each of the
different types of cysts and increases gradually within the next
days. An additional inspection within 1 hour after TRITC
treatment indicates that in the case of the stenoteles and holotrichous isorhizas all fluorescent cysts display irregularities in
their shapes if they are mounted in the high-osmotic PEG
solution (see Materials and Methods). Obviously, their full
intracapsular osmotic pressure is not yet established (Weber,
1989), and they must be considered as immature. This is also
true for a small fraction of the cysts at later times. For the
smaller atrichous isorhizas and desmonemes it is not clear
whether the few fluorescent cysts found immediately after
labelling (Fig. 5) are immature, but it is assumed that the
situation is similar to that in the other two cyst types.
Therefore, the ability of cysts to be labelled by TRITC is
lost at exactly the moment when the full internal osmotic
pressure is attained, i.e. the point at which the cysts become
operationally mature and have acquired their high buoyant
densities (Weber et al., 1987, 1988). The gradual increase in
the relative amounts of fluorescent nematocysts within a few
days, as illustrated in Fig. 5, supports the assumption that
following TRITC treatment all of the newly maturing nemato-
Fig. 4. Labelling of Hydra with TRITC. TRITC-labelled Hydra were fixed and mounted as outlined in Materials and Methods. The
fluorescence pictures shown in the upper row correspond to the respective Nomarski-DIC pictures of the lower row. (A) to (C) Selected parts of
the body column. The triangles indicate fluorescent late stenotele nematoblasts whereas asterisks mark a mature stenotele (A) and
nonfluorescent stenotele nematoblasts (C) (see text). (D) Cell-free nematocysts (s, stenotele; i, holotrichous isorhiza; d, desmonemes) isolated
from whole Hydra 5 days after TRITC-labelling. Bars, 20 µm (same magnification in (A) to C)).
Novel tools for studying nematocytes
AAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAA
AAA
AAAAAAAAAAAAAAAAA
AAA
50
Amount of labelled cysts in the whole polyp (%)
40
30
20
Stenoteles
Desmonemes
Isorhizas
10
0
0
20
40
60
Time after TRITC-labelling (hours)
80
100
80
60
40
20
cytes are fluorescent. As in the case of TROMI labelling, the
rates of increase of TRITC-labelled nematocytes with time can
vary considerably amongst different Hydra clones and even
within the same clones (see also below and Fig. 6).
The fate of TRITC-labelled nematoblasts
Dynamic aspects of the nematocyte turnover were studied by
analysing the body column and tentacles for the temporal
appearance of fluorescent cysts after labelling Hydra with
TRITC. Fig. 6 shows the time course of maturation of new (fluorescent) nematocytes and the displacement of old (non-fluorescent) ones in the body column (A) and the tentacles (B) of
the polyps. Since only mature cysts (plus a small fraction of
immature ones; see above) survive the preparation procedure
before counting, the figure confirms that the ability of stenoteles and isorhizas to become labelled is lost when the full intracapsular osmotic pressure is completely established. Since the
number of desmonemes in the body column is relatively low
and the fraction of presumably immature but morphologically
similar cells is rather high, the graph for the desmonemes is
shown artificially shifted to too-high absolute values.
As deduced from Fig. 6A new desmonemes replace old,
unlabelled ones in the body column with a t50% of about 0.5
day. The latter are assumed to migrate into the tentacles (see
above, and Bode and Flick, 1976). A t50% of about 1.0 day is
determined for the about 70 to 80% of stenoteles that are not
mounted in the body column (Table 1). The replacement
velocity of these latter ones, which are mounted and ready for
discharge, is much longer (t50% approx. 3 to 4 days; see Fig.
3). For the isorhizas the replacement rates cannot be estimated
precisely, but the steep initial increase indicates that the
replacement rates for the approx. 15% unmounted holotrichous
isorhizas (Table 1) and the relatively low amount of atrichous
isorhizas are probably similar to those of desmonemes.
Fig. 6B illustrates the situation in the tentacles of the same
polyps: labelled desmonemes and isorhizas migrate after maturation, apparently without any delay into tentacles, and their
number increases there at a nearly constant rate of approx. 15%
per day within the first 4 days. Stenoteles, in contrast, appear
only after a lag phase of approx. 1.0 to 1.5 days after matura-
A
0
Stenoteles
Desmonemes
Isorhizas
B
Amount of labelled cysts in the tentacles (%)
100
80
60
Fig. 5. Increase in labelled mature nematocysts after incubation of
Hydra with TRITC. After the indicated periods of time following
TRITC labelling, 20 whole polyps were frozen and used for the
isolation of their nematocysts. For desmonemes (triangles),
stenoteles (squares) and isorhizas (circles) the relative amount of
fluorescent cysts was determined by counting. Hydra were not fed
during this experiment. For details see Materials and Methods.
AAAAAAAAAA
AAAAAAAAAAAA
AAAAAAAAAA
AAAAAAAAAAAA
AAAAAAAAAAA
AAAAAAAAAA
AAAAAAAAAAAA
AAAAAAAAAAA
AAAAAAAAAA
AAAAAAAAAAA
AAA
AAAAAAAAAAA
AAA
AAAAAAAAAAA
AAA
AAAAAAAAAAA
AAAAAAAAA
AAAAAAAAAA
AAAAAAAAA
AAAAAAAAAA
AAAAAAAAA
AAAAAAAAAA
AAAAAAAAA
AAAAAAAAAA
AAAAAAAAAA
AAAAAAAAAA
Amount of labelled cysts in the body column (%)
100
40
20
0
409
F
F
F
0
1
2
4
3
5
Time after TRITC-labelling (days)
F
6
7
Fig. 6. Dynamic aspects of nematocyte maturation. To determine the
increase in newly maturated nematocytes, nematocysts were isolated
separately from body columns and from tentacles of Hydra 0 to 7
days after TRITC labelling and counted as described in the legend to
Fig. 5. Hydra were fed where indicated (F).
tion in tentacles. Thereafter their amount increases significantly faster than the amount of the other types of cysts.
The data of Fig. 6 agree with assumptions of Bode and Flick
(1976) that within about 9 days all the nematocytes of a
tentacle become replaced by new ones, due to loss of cells at
the distal end of the tentacles and proximal insertion of new
battery cells; the even more rapid increase in stenoteles
suggests that this type of cyst is used and therefore replaced
due to intense food capture at much higher rates than the
desmonemes and isorhizas.
In further experiments, both, the fundamental time course of
the graphs and the corresponding displacement rates, as well
as the typical lag phase for the appearance of stenoteles within
tentacles, were confirmed, although the absolute values may
differ to some degree.
The quantitative data of Fig. 6 could be sustained qualitatively by microscopic observation of nematocytes that
migrated through the body column of polyps into the tentacles.
For this purpose, distal parts of untreated polyps were grafted
onto proximal halves of Hydra 0, 1 and 2 days after incubation in TRITC.
Within the first day after labelling, high numbers of
desmonemes and isorhizas but essentially no stenoteles had
migrated to the unlabelled, distal part of the body column or
into tentacles. Atrichous isorhizas were the first nematocytes
to reach distal parts of the tentacles, followed by desmonemes
and holotrichous isorhizas. After two days, in addition,
numerous stenoteles were recorded in the upper body column
and within tentacles (up to more than 10 new stenoteles per
tentacle). This was observed both in transplants that had been
grafted immediately after labelling (2 days of parabiosis) and
in those grafted only one day after labelling (1 day of parabiosis). The pronounced increase in stenoteles migrating into
tentacles between day 2 and day 3, as demonstrated in Fig. 6,
could also be observed independently of the time of parabio-
410
J. Weber
sis in the same type of experiment (1 or 2 days of parabiosis).
These observations suggest that the delay of about one day for
stenoteles appearing in tentacles (Fig. 6B) is based on the
(apparent) lag-phase of stenotele development before starting
to migrate and not on a relatively slow average migration
velocity of these cells through distal (upper) parts of the body
column. Like other types of nematocytes, stenoteles, after this
lag phase, are able to migrate from lower parts of the body
column up to the tips of tentacles within less than 0.5 day.
In addition, it was found that the majority of desmonemes
and isorhizas are mounted most proximally in the new, still
empty battery cells of the tentacles as reported (Bode and Flick,
1976), whereas stenoteles are found more scattered along the
whole length of the tentacles. This confirms the assumption
(see above) that stenoteles discharge more frequently (and have
to be replaced by new ones more often) than other cysts.
DISCUSSION
It has been shown previously that multipotent stem cells of the
body column of Hydra that have been committed to nematocytes undergo three to five cycles of synchronous cell divisions
and develop into mature nematocytes within about 5 to 7 (for
desmonemes and isorhizas) or 7 to 8 (for stenoteles) days
(David and Gierer, 1974). During this differentiation period the
nematocysts grow within a modified Golgi vesicle via a highly
sophisticated cascade of assembly processes to their final size
and complex structure (Slautterback and Fawcett, 1959;
Holstein, 1981). The mature nematocytes migrate within the
ectodermal tissue to their final destination, the outer surface of
the body column or, more frequently, the ectoderm of the
tentacles. There the nematocytes are mounted and become susceptible to external stimuli that trigger their discharge (Watson
and Hessinger, 1989; Golz and Thurm, 1991). Although the
migration of nematocytes has been studied directly by microscopic observation in vivo (Günzl, 1971; Campbell and
Marcum, 1980) or in vitro (González Agosti and Stidwill,
1991, 1992) as well as statistically (mainly with radiolabelled
nematocytes) (Tardent and Morgenthaler, 1966; Zumstein,
1973; David and Gierer, 1974; Bode and Flick, 1976; Herlands
and Bode, 1974; Weber et al., 1978), many aspects of the
nematocyte movements remain unclear, and the overall
knowledge of the processes of nematoblast development, not
only in Hydra, is only marginal (see Campbell, 1988b; Kurz
et al., 1991).
The novel methods
The new cell dissociation method presented here is based on a
procedure for dissociating Hydra polyps into single cells,
which are able to reconstitute a new polyp (Gierer et al., 1972;
Flick and Bode, 1983), and on a similar protocol for dissociating marine cnidarians (Schmid et al., 1981). Compared to the
Gierer method, the one presented in this article allows a more
easy identification of the particular cell types, improved
intrinsic stability of the dissociated cells and almost complete
dissociation of body columns and tentacles without significant
amounts of cellular debris. It enables us to use the method for
quantitative work and to optimise the dissociation procedure
(changes in the buffer composition, dissociation temperature)
for particular purposes.
Compared to David’s (1973) maceration procedure, which
has succesfully been used mainly for quantitative analyses of
whole Hydra or tissue fractions including autoradiography, the
present dissociation method possesses the advantage of being
non-destructive; it still permits the identification and analysis
of subcellular structures.
In addition to the results presented in this article, dissociated
cells have succesfully been applied to flow cytometric cell
sorting (Maurer, 1988), various autoradiographic studies,
analyses of endocytotic events and the evaluation of selective
fluorescent staining of subcellular compartments (all unpublished).
There is good evidence that TROMI selectively labels those
nematocytes of living Hydra that are mounted and thus ready
to be triggered for discharge. This is demonstrated by microscopic inspection of labelled intact Hydra tissue and by quantitatively analysing the content and temporal decrease in fluorescent nematocytes from initially TROMI-labelled polyps.
Whereas dye that is covalently coupled to cnidocils can in
some cases be lost after regeneration of these fragile structures
(Golz and Thurm, 1990), the characteristic labelling of the rods
seems to persist for the rest of the nematocyte’s life span. The
fluorescence of these uncharacterised structural components of
the nematocyst’s enveloping cytoskeleton (Stidwill and
Honegger, 1989) can thus be considered as an indicator that
the particular nematocyte was mounted at the time of the
TROMI application. It is not clear why TROMI, which
generally seems to be unable to diffuse across cellular
membranes of Hydra, reacts preferentially with the intracellular rods. Our observation might indicate that the (apical) cell
membrane is in its physical properties somehow different
compared to other cell membranes facing the outer milieu, but
the preferences of the dye for the rods remain unexplained.
However, the high and selective affinities of TROMI for these
uncharacterized structural components of nematocytes could
now be used as a tool to investigate their biochemical nature
and physiological role.
As shown by microscopical observations in Hydra tissue
and by data obtained after isolation of intact nematocysts,
TRITC couples covalently (in addition to many other Hydra
tissue components) preferentially to cysts within nematoblasts
that are just about to become mature. In contrast to the reaction
with TROMI, here it is the cyst’s wall and the inverted tubule
and its associated structures that couple to the dye, but it is
again unclear what is the reason for this particular behaviour.
Since a similar labelling pattern is observed in permeabilised,
fixed Hydra, the labelling potency does not seem to be
governed by membrane permeabilities. It may rather depend
on temporal changes in the availability of reactive nucleophiles
of nematocysts during the phase of their final maturation. In
contrast to TROMI, TRITC is not a strict pulse-labelling agent
but stains late-stage nematoblasts during at least six days after
exposing the Hydra polyps to the dye, albeit at decreasing
intensities.
The staining properties of Hydra nematoblasts and nematocytes with TROMI and TRITC are not unique. It has been
found (unpublished) that there is a similar staining behaviour
with the pair of dyes N-(7-dimethylamino-4-methyl
coumarinyl)maleimide (Yamamoto et al., 1977) and monobromobimane (Kosower et al., 1979) and it is to be expected that
other dyes with similar properties can be found.
Novel tools for studying nematocytes
Turnover of nematocytes
The suitability of the present novel methods for studies of maturation, migration and turnover of nematocytes is demonstrated by the results obtained, which extend established data
(Zumstein, 1973; Bode and Flick, 1976). As outlined in Results
the different types of nematocytes lose their ability to be
stained by TRITC at the time when the final internal osmotic
pressure of their cysts is attained. In the following, it will be
considered as the time of becoming mature.
Desmonemes are normally not mounted in the body column
and, once mature immediately start to migrate to the tentacles.
Their mean time of residence in the body column as mature
cells is only approx. 0.5 day. Once in the tentacles the majority
of them are mounted proximally in newly differentiated battery
cells. Within about 4 days half of the tentacular desmoneme
population is replaced by new ones. Atrichous isorhizas, which
are rather difficult to observe because their number is low,
seem to behave similarly to desmonemes. Holotrichous
isorhizas are mounted mainly in the body column. They too
start to migrate immediately after the final maturation. Preliminary observations indicate that these nematocytes are not
mounted until 10 to 15 hours later. Holotrichous isorhizas that
migrate into tentacles again seem to behave similarly to
desmonemes.
Stenoteles, in contrast to the other three types of nematocytes, appear only after a lag phase of 1 to 1.5 days after maturation in the tentacles. Their turnover rate there, however, is
higher than either of the other types, presumably because they
are consumed in higher numbers than the others. As revealed
in preliminary observations, stenoteles are not mounted before
approx. 2 days after maturation.
Thus, while desmonemes and isorhizas start to migrate and
to be mounted immediately after maturation (with a certain
delay that includes the time to find the place, correct the
position and accomplish cell physiological changes), the
situation for stenoteles is different. The delay of 1 to 1.5 days
before stenoteles start to migrate could reflect an additional
maturation phase for these more complex types of nematocytes
or represent the ‘reservoir’ of mature stenoteles reported by
Zumstein (1973). Further experiments with Hydra that are
devoid of a considerable amount of stenoteles might clarify this
point.
Outlook
Studies of nematocyte movement and turnover, as e.g. shown
in Fig. 6, can now be performed without time-consuming
autoradiographic procedures in quick and straightforward
experiments. It will be possible after all, in conjunction with
the various well-established grafting techniques, to investigate
in detail the regulation of nematocyte growth and supply,
which are both obscure (Bode, 1988). In particular, assays that
allow the detection, quantification and eventually characterisation of stimulating, inhibitory and chemotactic factors influencing the differentiation, migration and mounting of the
different types of nematocytes can be established. In addition,
TRITC-labelled nematocytes can be recorded with time-lapse
video under fluorescence illumination for several hours
without considerable photo-bleaching; the direct observation
and analysis of migration of nematocytes in vivo (Campbell
and Marcum, 1980) will thus be simplified considerably. Fur-
411
thermore, the analysis of the maturation of Hydra nematocytes,
in particular the synthesis of the pressure-generating poly(γglutamic acid)s (Weber, 1989, 1990, 1994) and more detailed
quantitative studies on the turnover and mounting of nematocytes will be facilitated.
The method of TRITC labelling seems to be more generally
applicable. We have observed that in marine cnidarians (polyps
and medusae of Podocoryne, ephyra of Aurelia, acontia of Calliactis) some, presumably immature, populations of nematocytes/nematoblasts are preferentially stained and that mature
fluorescent nematocytes are found mounted later. In Hydra as
well as in the few other well-investigated hydrozoans it will be
of interest to study the turnover and migration of nematocytes
in relation to different recently discovered or postulated morphogenic factors (Fujisawa, 1987; Hassel and Berking, 1988;
Müller, 1989; Plickert, 1989; Lange et al., 1990; Lange and
Müller, 1991).
I thank Dr Pierre Tardent for his help and everlasting generous
support, Dr Marianne Klug and Dr Charles N. David for stimulating
discussions, and Dr Robert Stidwill for commenting on the manuscript. The work was supported financially by the Swiss National
Science Foundation (grant 31-29860.90).
REFERENCES
Blanquet, R. S. (1977). Cnidarian venoms. In Perspectives in Toxinology (ed.
A. W. Bernheimer), pp. 149-167. John Wiley & Sons, New York.
Blanquet, R. S. and Lenhoff, H. M. (1966). A disulfide-linked collagenous
protein of nematocyst capsules. Science 154, 152-153.
Bode, H., Berking, S., David, C. N., Gierer, A., Schaller, H. and Trenkner,
E. (1973). Quantitative analysis of cell types during growth and
morphogenesis in Hydra. Roux’s Arch. Dev. Biol. 171, 269-285.
Bode, H. R. and Flick, K. M. (1976). Distribution and dynamics of nematocyte
populations in Hydra attenuata. J. Cell Sci. 21, 15-34.
Bode, H. (1988). Control of nematocyte differentiation in Hydra. In The
Biology of Nematocysts (ed. D. A. Hessinger and H. M. Lenhoff), pp. 123142. Academic Press, San Diego.
Bosch, T. C. G. and David, C. N. (1991). Decision making in interstitial stem
cells of Hydra. In Vivo (Athen) 5, 515-520.
Burnett, J. W. and Calton, G. J. (1977). Review article: The chemistry and
toxicology of some venomous pelagic coelenterates. Toxicon 15, 177-196.
Burnett, J. W. and Calton, G. J. (1987). Venomous pelagic coelenterates:
chemistry, toxicology, immunology and treatment of their stings. Toxicon
25, 581-602.
Campbell, R. D. and Marcum, B. A. (1980). Nematocyte migration in Hydra:
evidence for contact guidance in vivo. J. Cell Sci. 41, 33-51.
Campbell, R. D. (1988a). Migration of nematocytes in hydrozoans. In The
Biology of Nematocysts (ed. D. A. Hessinger and H. M. Lenhoff), pp. 123142. Academic Press, San Diego.
Campbell, R. D. (1988b). The nematocyte: an encapsulation of developmental
processes. In The Biology of Nematocysts (ed. D. A. Hessinger and H. M.
Lenhoff), pp. 115-121. Academic Press, San Diego.
Campbell, R. D. (1989). Taxonomy of the European Hydra (Cnidaria,
Hydrozoa). A re-examination of its history with emphasis on the species
Hydra vulgaris Pallas, Hydra attenuata Pallas and Hydra circumcincta
Schulze. Zool. J. Linn. Soc. 95, 219-244.
David, C. N. (1973). A quantitative method for maceration of hydra tissue.
Roux’s Arch. Dev. Biol. 171, 259-268.
David, C. N. and Gierer, A. (1974). Cell cycle kinetics and development of
Hydra attenuata. III. Nerve and nematocyte differentiation. J. Cell Sci. 16,
359-375.
Drobnica, L., Kristian, P. and Augustin, J. (1977). The chemistry of the
-NCS group. In The Chemistry of Cyanates and Their Thio Derivatives (ed.
S. Patai), pp. 1003-1221. John Wiley & Sons, New York.
Flick, K. M. and Bode, H. R. (1983). Dissociating tissue into cells and the
development of Hydra from aggregated cells. In Hydra: Research Methods
(ed. H. M. Lenhoff), pp. 251-259. Plenum Press, New York.
412
J. Weber
Fujisawa, T. (1987). An endogenous inhibitor involved in position-dependent
stenotele differentiation in Hydra. Dev. Biol. 122, 210-216.
Gierer, A., Berking, S., Bode, H., David, C. N., Flick, K., Hansmann, G.,
Schaller, H. and Trenkner, E. (1972). Regeneration of Hydra from
reaggregated cells. Nature 239, 98-101.
Giloh, H. and Sedat, J. W. (1982). Fluorescence microscopy: reduced
photobleaching of rhodamine and fluorescein protein conjugates by n-propyl
gallate. Science 217, 1252-1255.
Golz, R. and Thurm, U. (1990). Cnidocil regeneration in nematocytes of
Hydra. Protoplasma 155, 95-105.
Golz, R. and Thurm, U. (1991). Cytoskeleton-membrane interactions in the
cnidocil complex of hydrozoan nematocytes. Cell Tiss. Res. 263, 573-583.
González Agosti, C. and Stidwill, R. P. (1991). In vitro migration of Hydra
nematocytes: the influence of the natural extracellular matrix (the mesoglea),
of collagen type IV and type I, laminin, and fibronectin on cell attachment,
migration parameters, and on patterns of cytoskeletal proteins. Cell Motil.
Cytoskel. 20, 215-227.
González Agosti, C. G. and Stidwill, R. P. (1992). The contributions of
microtubules and F-actin to the in-vitro migratory mechanisms of Hydra
nematocytes as determined by drug interference experiments. Exp. Cell Res.
200, 196-204.
Greber, M. J., David, C. N. and Holstein, T. W. (1992). A quantitative
method for seperation of living Hydra cells. Roux’s Arch. Dev. Biol. 201,
296-300.
Günzl, H. (1971). Dipurena reesi (Hydrozoa). Wanderung der Cnidoblasten in
den Rhizistolonen. In Encyclopaedia Cinematographica (ed. G. Wolf), pp.
3-15. Institut für den wissenschaftlichen Film, Göttingen, BRD.
Hassel, M. and Berking, S. (1988). Nerve cell and nematocyte production in
Hydra is deregulated by lithium ions. Roux’s Arch. Dev. Biol. 197, 491-495.
Herlands, R. L. and Bode, H. R. (1974). Oriented migration of interstitial cells
and nematocytes in Hydra attenuata. Roux’s Arch. Dev. Biol. 176, 67-88.
Hessinger, D. A. and Lenhoff, H. M. (1988). The Biology of Nematocysts.
Academic Press, San Diego.
Holstein, T. (1981). The morphogenesis of nematocytes in Hydra and
Forskalia: an ultrastructural study. J. Ultrastruct. Res. 75, 276-290.
Klimek, F. (1979). Untersuchungen zur Separation und Aggregation von
Hydrazellen. Ph.D. thesis, Tübingen, BRD.
Kosower, N. S., Kosower, E. M., Newton, G. L. and Ranney, H. M. (1979).
Bimane fluorescent labels: labeling of normal human red cells under
physiological conditions. Proc. Nat. Acad. Sci. 76, 3382-3386.
Kurz, E. M., Holstein, T. W., Petri, B. M., Engel, J. and David, C. N. (1991).
Mini-collagens in Hydra nematocysts. J. Cell Biol. 115, 1159-1169.
Lange, R. G., Holzenburg, P. and Müller, W. A. (1990). Pulses of ammonia
and methylamine induce down-regulation of nematocyte and nerve cell
populations in Hydrozoa (Hydra; Hydractinia). Roux’s Arch. Dev. Biol. 199,
123-133.
Lange, R. G. and Müller, W. A. (1991). SIF, a novel morphogenic inducer in
Hydrozoa. Dev. Biol. 147, 121-132.
Loomis, W. F. and Lenhoff, H. M. (1956). Growth and sexual differentiation
of Hydra mass culture. J. Exp. Zool. 132, 555-574.
Mariscal, R. N. (1974). Nematocysts. In Coelenterate Biology (ed. L.
Muscatine and H. M. Lenhoff), pp. 129-178. Academic Press, New York.
Mariscal, R. N. (1984). Cnidaria: Cnidae. In Biology of the Integument, vol. 1,
Invertebrates (ed. J. Bereiter-Hahn, A. G. Matoltsy and K. S. Richards), pp.
57-68. Springer Verlag, Berlin.
Maurer, A. (1988). Investigations about the localization of the head activator
in Hydra attenuata Pall. (Hydrozoa, Cnidaria). Ph.D. thesis, University of
Zurich, Switzerland.
Müller, A. W. (1989). Diacylglycerol-induced multihead formation in Hydra.
Development 105, 309-316.
Plickert, G. (1989). Proportion-altering factor (PAF) stimulates nerve cell
formation in Hydractinia echinata. Cell Differ. Dev. 26, 19-28.
Schmid, V., Stidwill, R., Bally, A., Marcum, B. and Tardent, P. (1981). Heat
dissociation and maceration of marine Cnidaria. Roux’s Arch. Dev. Biol. 190,
143-149.
Slautterback, D., B. and Fawcett, D. W. (1959). The development of the
cnidoblast in Hydra. An electron microscope study of cell differentiation. J.
Biophys. Biochem. Cytol. 5, 441-452.
Stidwill, R. P. and Honegger, T. G. (1989). A single layer of microtubules is
part of a complex cytoskeleton in mature nematocytes of Hydra. Tissue &
Cell 21, 179-188.
Tardent, P. (1966). Zur Sexualbiologie von Hydra attenuata Pall. Rev. Suisse
Zool. 73, 357-381.
Tardent, P. and Morgenthaler, U. (1966). Autoradiographische
Untersuchungen zum Problem der Zellwanderungen bei Hydra attenuata
Pall. Rev. Suisse Zool. 78, 468-480.
Tardent, P., Holstein, T., Weber, J. and Klug, M. (1985). The
morphodynamics and actions of stenotele nematocysts in Hydra. Arch. Sci.
Genève 38, 401-418.
Watson, M. G. and Hessinger, D. A. (1989). Cnidocyte mechanoreceptors are
tuned to the movements of swimming prey by chemoreceptors. Science 243,
1589-1591.
Weber, G., Honegger, T. and Tardent, P. (1978). Neuorientierung der
Nesselzellwanderung bei Hydra attenuata Pall. durch transplantierte
Tentakel. Rev. Suisse Zool. 85, 768-774.
Weber, J., Klug, M. and Tardent, P. (1987). Some physical and chemical
properties of purified nematocysts of Hydra attenuata Pall (Hydrozoa,
Cnidaria). Comp. Biochem. Physiol. 88B, 855-862.
Weber, J., Klug, M. and Tardent, P. (1988). Chemistry of hydra nematocysts.
In The Biology of Nematocysts (ed. D. A. Hessinger and H. M. Lenhoff), pp.
427-444. Academic Press, San Diego.
Weber, J. (1989). Nematocysts (stinging capsules of Cnidaria) as Donnanpotential-dominated osmotic systems. Eur. J. Biochem. 184, 465-476.
Weber, J. (1990). Poly(γ-glutamic acid)s are the major constituents of
nematocysts in Hydra (Hydrozoa, Cnidaria). J. Biol. Chem. 265, 9664-9669.
Weber, J. (1994). The metabolism of poly(γ-glutamic acid)s of nematocysts in
Hydra vulgaris: detection of two distinct hydrolytic enzymes in endoderm
and in nematocysts. Comp. Biochem. Physiol. 107B, 21-32.
Williams, R. B., Cornelius, P. F. S., Hughes, R. G. and Robson, E. A. (1991).
Coelenterate Biology: Recent research on Cnidaria and Ctenophora.
Proceedings of the Fifth International Conference on Coelenterate Biology,
1989. In Developments in Hydrobiology, vol. 66 (ed. H. J. Dumont), pp. 0742. Kluwer Academic Publ., Dordrecht, NE.
Yamamoto, K., Sekine, T. and Kanaoka, Y. (1977). Fluorescent thiol
reagents XII. Fluorescent tracer method for protein SH groups using N-(7dimethylamino-4-methyl coumarinyl) maleimide. An application to the
proteins separated by SDS-polyacrylamide gel electrophoresis. Anal.
Biochem. 79, 83-94.
Zumstein, A. (1973). Regulation der Nematocyten-Produktion bei Hydra
attenuata Pall. Roux’s Arch. Dev. Biol. 173, 294-318.
(Received 28 March 1994 - Accepted 24 August 1994)