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First published online February 15, 2008
Journal of Experimental Biology 211, 731-740 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.011148
Physiological characterization of stolon regression in a colonial hydroid
Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115, USA
* Author for correspondence (e-mail: neilb{at}niu.edu)
Accepted 24 December 2007
 |
Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: cell death, clonal organism, cnidarian, evolution and development, evolutionary morphology, hydroid, Podocoryna, Podocoryne, reactive oxygen species, reactive nitrogen species
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
van Kleunen and Fisher, 2001).
In these clonal groups, the morphology can be idealized as comprising feeding
and reproductive units, here termed polyps, which are interconnected by
vascular stolons. Runner-like forms show widely spaced polyps and long
stolonal connections, whereas sheet-like forms show closely packed polyps with
short stolonal connections (Buss and
Blackstone, 1991). These different morphological patterns may
reflect changes in the timing of the production of polyps and stolon tips
relative to rates of stolon growth and colony maturation; high ratios of
production yield sheets, and low ratios yield runners.
Colonial hydroids can be particularly useful for investigating the
differences between runners and sheets. These hydroids are representatives of
cnidarians, a group of early-evolving animals. Considerable research has
focused on the mechanisms underlying their development. Early studies
concentrated on physiological parameters, particularly gastrovascular flow.
For instance, Hale (Hale,
1964) described the growth of stolons in terms of the influence of
gastrovascular flow on the cyclic pattern of forward surges and partial
backward retraction in the stolon tip. Wyttenbach
(Wyttenbach, 1968;
Wyttenbach, 1969;
Wyttenbach, 1973) further
characterized this type of stolon elongation in terms of colony-level
variations in growth rate, cycle phases, and features of gastrovascular flow.
Beloussov and collaborators (e.g.
Beloussov, 1973;
Beloussov et al., 1989) also
studied stolon tip extension, but from the perspective of cell-level
morphogenesis and physiology. Schierwater et al.
(Schierwater et al., 1992)
provided an overview of the various physiological parameters that contribute
to gastrovascular flow and proposed a cellular mechanism for stolonal
expansion and contraction. Dudgeon and Buss
(Dudgeon and Buss, 1996) also
emphasized the importance of gastrovascular flow in colony development. At the
same time, a variety of studies focused on the effects of morphogens on colony
growth (Plickert et al., 1987)
including head activator (Schaller et al.,
1989), stolon-inducing factor
(Lange and Müller, 1991)
and other peptides (Takahashi et al.,
1997). More recent studies have begun to elucidate the role of
gene activity in colony development. For instance, Cartwright and Buss
(Cartwright and Buss, 1999)
and Cartwright et al. (Cartwright et al.,
1999; Cartwright et al.,
2006) explored the expression of a well-studied parahox gene in
the various components of the colony whose frequencies and distributions
contribute to the overall pattern.
Despite these and other efforts to understand colony development, the
relevance of regressive processes has not yet been considered in detail. For
instance, stolon regression could influence both runner- and sheet-like
patterns: the former could be the result of little regression of peripheral
stolons, whereas the latter could be the result of greater amounts of such
regression. Increasingly, developmental biologists are recognizing the
widespread occurrence and importance of tissue regression and related
processes and their contribution to pattern formation. For example, tissue
regression plays a role in sculpting the individual digits of the limbs of
vertebrates (Zuzarte-Luís and
Hurlé, 2002) and the segments of the body of some
invertebrates (Lohmann et al.,
2002). The regression of the tadpole's tail during its
metamorphosis into a frog is another well-known example
(Tata, 2006). Tissue
regression is also important in the transition of insects from their larval
forms to their adult forms and has been described in a variety of species
(Hori et al., 2000).
|
;
Blackstone et al., 2005).
Thus, stolon regression may be an active process involving other active
processes such as some form of regulated cell death. In this context, we
developed a reliable method of pharmacologically inducing stolon regression in
a colonial hydroid and have attempted to characterize this process at
morphological, physiological and cellular levels. |
MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). The clone, designated as P3, has been used in previous
studies for over 14 years. All experiments were performed on clonal replicates
grown from single polyp explants of a source colony. For each experiment,
control and treated colonies were explanted from the same source colony to
minimize non-genetic sources of variation
(Ponczek and Blackstone,
2001). For measures of ROS, reactive nitrogen species (RNS),
gastrovascular flow and cell death, colonies were grown on 15 mm diameter
round glass coverslips. For transmission electron microscopy (TEM), clonal
replicates were grown on a piece of clear polyethylene tied to a glass slide.
All experiments were carried out at 20.5°C.
Treatment with hydrogen peroxide and nitric oxide
For characterization of stolon regression, it was deemed necessary to
develop a protocol that reliably triggered the regression of a large number of
stolons in a single colony. Since vitamin C-treated colonies typically show a
variable response, colonies were treated with hydrogen peroxide
(H2O2) or a nitric oxide donor,
S-nitroso-N-acetyl-penicillamine (SNAP). For all H2O2
experiments, colonies were treated in a 5 mmol l–1 solution
of H2O2 and seawater
(Vernole et al., 1998) with
controls placed in plain seawater. For all NO experiments, colonies were
treated in seawater with SNAP dissolved in dimethyl sulfoxide (DMSO) at a
final concentration of 0.5 mmol l–1. Controls were treated
with an equivalent concentration of DMSO. For both H2O2
and NO experiments, a set of 6–7 treated and 6–7 control colonies
were incubated for 
60 min beginning 24 h after feeding. Following
treatment in respective solutions, the colonies were incubated in plain
seawater and staining solution, typically for 60 min prior to imaging or
fixation. Excluding colonies analyzed for several of the cell death assays and
for temporal sequence of events, fixation or imaging typically was done 2 h
after treatment began.
Measurement of reactive oxygen species
The compound 2',7'-dichlorodihydrofluorescein diacetate
(H2DCFDA; Molecular Probes, Eugene, OR, USA) was used as a
semi-quantitative assay of general oxidative stress
(Jantzen et al., 1998). This
non-fluorescent dye is taken up by cells, and the acetate groups are removed
by intracellular esterases to form H2DCF. Oxidation within cells
leads to the fluorescent product, which can then be visualized using
fluorescence microscopy. Molecules that oxidize H2DCF will
typically oxidize cysteine residues, histidine residues and iron-sulfur
clusters of proteins. The oxidation of these residues and clusters are among
the primary mechanisms by which ROS can affect signaling pathways
(Armstrong et al., 2004;
Filomeni et al., 2005;
Lee and Helmann, 2006;
Salmeen et al., 2003;
van Montfort et al., 2003).
Thus, while there may be some debate as to whether the activation of
H2DCF is specific for the detection of H2O2
(Finkel, 2001), this assay
quantifies the extent to which potential signaling pathways may be affected. A
10 mmol l–1 stock solution of H2DCFDA was prepared
in anhydrous DMSO. After incubation in SNAP or H2O2,
treatment solutions were replaced with plain seawater and H2DCFDA
was added to each dish to a concentration of 10 µmol l–1.
Colonies were then incubated in the dark for 60 min. Subsequently, each
colony was imaged in a microscope chamber containing plain seawater using an
Orca-100 camera (Hamamatsu Photonics, Hamamatsu City, Japan) and an Axiovert
135 microscope (Carl Zeiss, Jena, Germany). ROS-related oxidative processes
(as indicated by H2DCFDA-derived dichlorofluorescein) were imaged
for three stolon tips per colony (excitation 450–490 nm, emission
515–565 nm). The luminance and area of each stolon tip was measured in
Image-Pro Plus (Media Cybernetics, Silver Spring, MD, USA) by: (1) using a
bright-field image of the stolon tip to define the area of the stolon and the
area of the background; (2) with these areas serving as `areas of interest'
for the fluorescent image, allowing the software to measure average luminance
of both areas; and (3) exporting these measurements to file. The area of each
stolon was thus defined, and the luminance of the stolon was adjusted for the
background luminance by subtraction. These measures were analyzed by a nested
analysis of variance (stolon tips within clonal replicates, clonal replicates
within treatments).
Measurement of reactive nitrogen species
The compound 4-amino-5-methylamino-2',7'-difluorofluorescein
(DAF-FM) diacetate (Molecular Probes, Eugene, Oregon, USA) was used as a
semi-quantitative indicator of low concentrations of NO
(Kojima et al., 1999). This
non-fluorescent dye is taken up by cells, and the acetate groups are removed
by intracellular esterases to form DAF-FM. Upon reaction with NO, DAF-FM forms
a fluorescent benzotriazole derivative. A 2 mmol l–1 stock
solution of DAF-FM diacetate was prepared in anhydrous DMSO. After incubation
of colonies in SNAP or H2O2, treatment solutions were
replaced with plain seawater, and DAF-FM diacetate stock solution was added to
each dish to a concentration of 2 µmol l–1. Colonies were
then incubated for 60 min in the dark prior to measurement. Images were
obtained (excitation 450–490 nm, emission 515–565 nm), measured
and analyzed as described above.
Cell death analyses
The similar effects of H2O2 and SNAP on stolon
regression (see Results), as well as clear parallels to the effects of
treatment with vitamin C (Blackstone et
al., 2004a; Blackstone et al.,
2005), suggest that a similar process or processes is occurring
with all three treatments. For simplicity, subsequent characterization of
stolon regression thus focused on treatment with H2O2.
To determine the extent of cell death in stolon regression, several standard
assays were carried out (Costa-Pereira and
Cotter, 1999; Frey,
1995; van Engeland et al.,
1999; Wilson and Potten,
1999). The assays and kits used were chosen specifically for their
ability to distinguish between live cells, necrotic cells and apoptotic cells.
The Live–Dead Cell Staining Kit (BioVision Research Products,
Mountainview, California, USA) was used on H2O2-treated
and control colonies following the manufacturer's protocol except that
seawater was substituted for staining buffer and incubation was at 20.5°C
instead of 37°C. To detect phosphatidylserine, which is exposed during
cell death, Annexin V-FITC Apoptosis Detection Kit (BioVision Research
Products, Mountainview, CA, USA) was used on control colonies as well as
colonies treated with H2O2. Finally, the loss of
mitochondrial transmembrane potential is a characteristic of some forms of
cell death. Mitochondrial membrane potential can be indicated by the cationic
dye
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine
iodide. None of these in vivo assays produced reliable results for
the stolon system of colonial hydroids. Other assays (e.g. DNA laddering) were
deemed inappropriate because they are not localizable to stolon tips.
For comparisons of DNA fragmentation, the TUNEL (terminal deoxynucleotide
transferase-mediated dUTP-biotin nick-end labeling) detection kit, ApoDIRECT
In Situ DNA Fragmentation Assay Kit (BioVision Research Products,
Mountainview, CA, USA), was used (van
Engeland et al., 1999; Wilson
and Potten, 1999). After incubation of colonies in
H2O2, treatment solutions were replaced with plain
seawater and colonies were incubated for an additional 60 min to mimic
methods used for assaying ROS and RNS. Colonies (treated and controls) were
then fixed and stained following the ApoDIRECT Assay protocol with slight
modifications for adherent cells. Washing steps were performed in Petri dishes
using 50 ml of wash buffer for each washing step. After the staining solution
was prepared, the colonies were transferred to dry Petri dishes and the
staining solution was pipetted onto the colonies. Rinsing steps were performed
in Petri dishes using 50 ml of rinse buffer for each rinsing step. Following
rinsing, the colonies were placed in 25 ml of propidium iodide (PI)/RNase
staining buffer. Fragmented DNA was labeled using terminal deoxynucleotidyl
transferase to catalyze the incorporation of fluorescein-12-dUTP at the free
3'-hydroxyl ends of the DNA. The stolon cells containing fragmented DNA
were visualized using fluorescence microscopy (excitation 450–490 nm,
emission 515–565 nm). All stolon cells (regardless of whether or not
they contained fragmented DNA) were counterstained using the PI/RNase solution
and were visualized using fluorescence microscopy (excitation 546 nm, emission
>590 nm). Images were obtained, measured and analyzed as described
above.
Comparisons of stolon tip structure
Stolons of control colonies and colonies treated with
H2O2 were examined using transmission electron
microscopy (TEM). Following an 60 min treatment and a subsequent 60
min incubation in plain seawater, colonies were fixed in 2.5% glutaraldehyde
for 3 h at 4°C followed by three 10 min rinses in Millonig's phosphate
buffer. Specimens were postfixed in 1% osmium tetroxide for 2 h at room
temperature followed by three 10 min rinses in Millonig's phosphate buffer,
then dehydrated in an ethanol series and cleared in acetone. Colonies were
infiltrated and embedded in EMbed 812 resin (Electron Microscopy Sciences,
Hatfield, PA, USA) and sectioned on a Reichert OmU2 ultramicrotome using a
diatome diamond knife. Sections of approximately 90 nm in thickness were
collected on formvar-coated slot grids or 75-mesh copper grids. Sections were
stained with uranyl acetate for 20 min and with lead citrate for 40 min.
Subsequently, sections were examined using a Hitachi H-600 transmission
electron microscope. Micrographs of specimens were obtained using Kodak 4489
electron microscope film and negatives were then scanned.
Comparisons of gastrovascular flow
Videos of stolons lasting 10 min were taken at a point 250 µm
behind the tip of a stolon using the Axiovert microscope and a Dage MTI-72
camera. Three stolon tips per colony were videotaped. A total of five
H2O2-treated colonies and five control colonies were
treated 30 min after feeding for 60 min and were imaged in plain
seawater 90–180 min after feeding and 10–100 min after treatment.
Using Image-Pro Plus, 100 images were extracted from each video at 5 s
intervals. Stolon width (perisarc to perisarc) and lumen width (endoderm to
endoderm) were measured for each image. Measurements of maximum and minimum
lumen width were obtained for three consecutive cycles. The period (in s) of
each cycle was also measured. The rate of gastrovascular flow can be estimated
as lumen width (maximum – minimum) divided by cycle period and stolon
width (Blackstone, 1996).
Although the two-dimensional analysis of flow requires certain assumptions
(e.g. Bagatto and Burggren,
2006), these assumptions seemed justifiable in this
experiment.
Temporal sequence of stolon regression
To provide a better understanding of the temporal sequence of events during
stolon regression, two sets of observations were carried out. First, an entire
colony was treated with H2O2 for 1 h and observed using
a dissecting microscope during and after treatment for >72 h. Second, eight
stolon tips from eight different colonies were observed. Each colony was
placed into a seawater-containing chamber for the Zeiss inverted microscope
and videotaped prior to treatment for 5 min. H2O2 was
then added to the chamber to a concentration of 5 mmol l–1.
Colonies were continually taped until regression appeared to stop, which took
anywhere from 30 to 90 min. Using Image-Pro Plus, images were extracted from
each video at 30 s intervals. This time-lapse sequence was then examined and
the timing of events associated with stolon regression was noted.
|
RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Blackstone et al., 2005),
treatments with H2O2 and NO donors typically cause
regression of peripheral stolons in P. carnea colonies as well as a
flux of ROS and RNS (Figs 2,
3). Of 63 stolons measured from
control colonies in the peroxide experiments, only one exhibited regression.
Of 63 stolons measured from treated colonies in the peroxide experiments, 62
exhibited regression (comparing treated to controls, 
2=118,
d.f.=1, P 
2=27, d.f.=1,
P
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Cell death analyses
Regressive processes in development frequently involve cell death. Many of
the standard in vivo assays for cell death
(Costa-Pereira and Cotter,
1999; Frey, 1995;
van Engeland et al., 1999;
Wilson and Potten, 1999) do
not work well in the hydroid stolon system and yield variable and unreliable
staining (data not shown). It is probable that the molecules involved in these
assays do not reliably enter an intact stolon. To detect patterns of DNA
fragmentation, TUNEL assays (in which stolons are fixed and permeabilized) did
show a reliable pattern of staining (Fig.
5). In H2O2-treated colonies, the TUNEL
fluorescent images showed a strong between-treatment difference
(Fig. 6A; F=119,
d.f.=1, 12, P
;
Blanco et al., 2005;
Mourdjeva et al., 2005). Note
that some assays (e.g. DNA laddering) were deemed inappropriate because they
cannot be restricted to stolon tips.
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Comparisons of stolon tip structure
Ultrastructural features of healthy hydroid epitheliomuscular cells (EMCs)
include a general columnar shape with one or more large vacuoles often
surrounding the nucleus (Thomas and
Edwards, 1991). Myoid processes, the contractile basal extensions
of EMCs, are typically undetectable in stolon EMCs
(Schierwater et al., 1992).
Control and H2O2-treated stolons showed clear
ultrastructural differences when visualized with TEM (Figs
7,
8). EMCs in control stolons had
the characteristic columnar shape, typically with the cell nucleus suspended
in a large vacuole (Fig.
7A–D) (Blackstone et
al., 2004b), whereas the stolon tip maintained a smooth, rounded
appearance (Fig. 7E). Treated
stolons were often contracted (as indicated by the space between the ectoderm
and the perisarc) and the cells showed extensive damage
(Fig. 8A–E). The
differences between cells of treated and control stolons cannot be explained
entirely by contraction state of the stolon (e.g. the amount of stolon
contraction in Fig. 7D and
Fig. 8A appears approximately
similar, but the cells look very different). Images show some recognizable
features of cell death. Some of these features are characteristic of necrosis,
such as flocculent condensation of chromatin
(Fig. 8A,B) and vacuolation of
the cytoplasm (Fig. 8A–E)
(Syntichaki and Tavernarakis,
2003). Other features seem to be more characteristic of apoptosis,
such as the lack of modification to cytoplasmic organelles such as
mitochondria (Fig. 8C)
(Syntichaki and Tavernarakis,
2003; Galluzzi et al.,
2007). Compaction of the nucleus
(Fig. 8A,B,D) and fragmentation
of the plasma membrane into numerous small vesicles
(Fig. 8A,E) also suggest
apoptosis (Syntichaki and Tavernarakis,
2003; Galluzzi et al.,
2007). Again, in other studies, features of regulated cell death
(e.g. DNA fragmentation, morphological changes, caspase activity) have been
detected over a similar time course
(Matsura et al., 2002;
Blanco et al., 2005;
Mourdjeva et al., 2005). Not
all of these features are found in all cells of treated stolons. However, none
of these features has been found in any cells of non-regressing, control
stolons. Indeed, Kroemer et al. (Kroemer
et al., 2005) stress that it "must be remembered that dying
in a cell population is not a synchronous but rather a stochastic process, and
that at a given time, individual cells will be at different stages of the
dying process." Thus, according to the recommendations of the
Nomenclature Committee on Cell Death, cells of treated stolons exhibited five
of the eight morphological features that define apoptosis, one of the five
features that define necrosis, and none of the features used to define
autophagy (Kroemer et al.,
2005).
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), this observation is consistent with the idea that
gastrovascular flow is the proximate trigger of regression.
Close observation of stolon tips of eight colonies indicate that stolon
regression follows a relatively stereotyped sequence of events. After
treatment with H2O2, the first step of stolon regression
involves the cessation of gastrovascular flow. In seven of the eight colonies
observed, this occurred within 0.5–2.0 min following treatment (13 min
for the outlier). Direct observations of gastrovascular flow to tips suggest a
somewhat quicker cessation of flow than whole-colony observations, possibly
because (1) gastrovascular flow can be better observed at high magnification,
or (2) flow to stolon tips ceases slightly before flow throughout the colony,
or both. Shortly thereafter, the stolons begin to regress. In seven of the
eight colonies observed, regression began 1.0–4.5 min after the
initiation of treatment (49 min for the outlier). Finally, in seven of the
eight colonies observed stolonal regression ceased within 8–22.5 min
after treatment (90 min for the outlier). At this point, the stolon tip still
appeared healthy. In some cases, regressing tips displayed a much more
distorted appearance (e.g. Fig.
2G). This distortion featured a misshapen tip, often leaving small
clusters of cells `stranded' following regression (e.g.
Fig. 2C). The almost immediate
cessation of gastrovascular flow following treatment suggests that this event
is the proximate trigger of stolon regression. Regression may thus be part of
the regulation of a hydroid colony by `self inspection'
(Buss, 2001). Resolving the
timing of the buildup of endogenous ROS and RNS relative to the cessation of
gastrovascular flow proved difficult; for instance, the intense excitation
wavelengths generated by the mercury-arc lamp of the Axiovert microscope may,
by itself, affect gastrovascular flow to the stolon tip. Nevertheless,
observations of numerous stolon tips (e.g. Figs
2 and
3) suggest that accumulation of
endogenous ROS and RNS begins shortly after gastrovascular flow ceases.
|
DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Blackstone et al., 2005). For
detailed studies, many stolons of a colony can be reliably induced to regress
by treatments with peroxide or nitric oxide. This process begins with a
cessation of gastrovascular flow to the distal part of the stolon. High levels
of endogenous ROS and RNS then accumulate as the stolon begins to regress.
Remarkably, exogenous treatments with either H2O2 or a
NO donor equivalently trigger endogenous formation of both
H2O2 and NO. Transmission electron microscopy reveals
that regressing stolons have atypical ultrastructural features. Cell death
during regression is suggested by both TUNEL and TEM.
These results further support previous work suggesting a potentially large
role for ROS and RNS in cnidarian signaling. Several examples will be briefly
described to provide an appreciation of both the species and the mechanistic
diversity of such signaling. First, Perez and Weis
(Perez and Weis, 2006) suggest
that symbiotic algae in heat-stressed anthozoans release ROS, which in turn
trigger the release of NO from the host cells. By-products of this
ROS–RNS signaling may contribute to anthozoan bleaching, that is, the
release of the symbiotic algae. Second, Berking et al.
(Berking et al., 2005) suggest
that in some scyphozoans metabolically produced peroxides oxidize iodide to
iodine. Endogenous tyrosine reacts with iodine to produce iodiferous tyrosine
compounds. This process may trigger medusa formation, provide an oxidant
defense system, and perhaps even supply the evolutionary roots of the
vertebrate hormone thyroxin. Finally, building on earlier work, Doolen et al.
(Doolen et al., 2007) suggest
that ROS may have a crucial role in runner-like (moderate ROS) or sheet-like
(low ROS) growth in P. carnea. The current research extends these
findings by showing that, whereas moderate levels of ROS may lead to rapid
colony growth and a runner-like form, high levels of ROS are involved in
another process – stolon regression – which in turn may lead to
more sheet-like growth. The taxonomic diversity of these results – in
anthozoans, scyphozoans and hydrozoans – is matched by the diversity of
pathways – host–symbiont interactions, medusa formation and colony
development. The `many pathways' view of ROS and RNS signaling in cnidarians
is further supported by Blackstone et al.
(Blackstone et al., 2005).
Regulated cell death is increasingly accorded a prominent role in many
biological processes, including those that involve tissue regression. Although
programmed cell death is often equated with apoptosis, it is now clear that
some forms of active or regulated cell death simply do not meet the criteria
for apoptosis and that apoptosis is but one form of programmed or regulated
cell death (e.g. Bredesen et al.,
2006; Galluzzi et al.,
2007). Thus, a plurality of cell death pathways and features is
emerging (Sperandio et al.,
2000; Proskuryakov et al.,
2003; Syntichaki and
Tavernarakis, 2003;
Guimarães and Linden,
2004; Bredesen et al.,
2006; Skulachev,
2006; Galluzzi et al.,
2007). Regulated forms of cell death are widespread in cnidarians,
and considerable work has been done to characterize these processes. In the
hydrozoan Hydra, cell death regulates growth
(David et al., 2005), disposes
of nurse cells during oogenesis (Technau
et al., 2003; Alexandrova et
al., 2005), and eliminates allogeneic cells
(Kuznetsov et al., 2002). In
the hydrozoan Hydractinia, cell death facilitates metamorphosis
(Seipp et al., 2001;
Seipp et al., 2006). In
anthozoans, cell death has a role in fission
(Mire, 1998;
Geller et al., 2005) and in
bleaching (Dunn et al., 2002;
Perez and Weis, 2006).
It is not clear whether or in what form cell death occurs during stolon
regression. As noted in the Results, the recommendations of the Nomenclature
Committee on Cell Death suggest that at least some of the features of
apoptosis are found in stolon cells of treated colonies
(Kroemer et al., 2005). The
pulse of endogenous ROS/RNS coincident with stolon regression also suggests an
active process. Certainly, other studies have documented an association
between cell death pathways and ROS/RNS, both in general (e.g.
Lin and Beal, 2006) and in
other cnidarians (Perez and Weiss,
2006). At this point, however, conclusions that are more
definitive are not possible and we would suggest that any particular form of
cell death cannot be ruled in or out with the results provided. Given the
constraints of the hydroid stolon system, a useful next step may be to
determine the effects of inhibitors of caspases. Caspases are generally
considered the effector enzymes for apoptosis
(Cikala et al., 1999;
Seipp et al., 2006) (but see
Galluzzi et al., 2007).
Investigations of this nature should provide further useful insights into the
relationship between ROS/RNS, cell death and stolon regression in the
development of colonial hydroids.
LIST OF ABBREVIATIONS
|
Acknowledgments |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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