Redox signaling in the growth and development of colonial hydroids
Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115, USA
(e-mail: neilb{at}niu.edu)
Accepted 6 November 2002
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Summary |
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Key words: clonal, colony development, evolutionary morphology, hydroid, Podocoryna, Podocoryne, reactive oxygen species, redox signaling
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Introduction |
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Redox signaling is a reliable mechanism by which an environmental signal
can be transduced into gene activity
(Allen, 1993). In animals,
redox signaling typically occurs when the redox states of electron carriers of
the mitochondria are perturbed by substrate
(Nishikawa et al., 2000
;
Blackstone, 2001
;
Brownlee, 2001
;
Rutter et al., 2001
) or
related factors (Bürkle,
2000
; Coffman and Davidson,
2001
; Larsen and Clarke,
2002
). Such perturbations can alter the rate of formation of
reactive oxygen species (ROS; reduced electron carriers are more likely to
donate electrons to oxygen, while oxidized electron carriers are less likely
to do so). ROS are frequently, but not always, a key intermediary in metabolic
and redox signaling (Nishikawa et al.,
2000
; Brownlee,
2001
; Echtay et al.,
2002
; Larsen and Clarke,
2002
; Nemoto and Finkel,
2002
). Such a mechanism may function in colonial animals. For
instance, if a growing hydroid colony encounters an area locally rich in food,
polyps in the food-rich area will experience a surfeit of nutrients. These
nutrients will trigger contractions of polyp epitheliomuscular cells and
resulting gastrovascular flow (Wagner et
al., 1998
; Dudgeon et al.,
1999
). Because of this metabolic demand, fed polyps will be more
oxidized, with lower levels of peroxide, than unfed polyps
(Blackstone, 2001
). If such a
redox gradient can differentially affect the timing of polyp and stolon tip
development, adaptive changes in the local pattern of colony development can
result.
This hypothesis allows a series of predictions about the effects of
commonly used experimental manipulations of the mitochondrial electron
transport chain with uncouplers of oxidative phosphorylation or inhibitors of
the individual complexes of this chain
(Scheffler, 1999). The major
sites of ROS formation are found at NADH dehydrogenase of complex I and at the
interface between coenzyme Q and complex III
(Nishikawa et al., 2000
).
Inhibitors of complexes III and IV should thus upregulate ROS from both sites;
inhibitors of complex I that act `downstream' of NADH dehydrogenase should
upregulate ROS from the first but not the second site, while uncouplers of
oxidative phosphorylation should downregulate ROS from both sites
(Fig. 1). If ROS mediate colony
development, inhibitors of complexes III and IV should produce similar
phenotypic effects, and these effects should differ from those of uncouplers
of oxidative phosphorylation. Other things being equal, inhibitors of complex
I should have intermediate phenotypic effects. Treatments of colonial hydroids
with azide (an inhibitor of complex IV) and dinitrophenol (an uncoupler of
oxidative phosphorylation) support this hypothesis; the former leads to
relatively reduced redox states and runner-like growth, while the latter leads
to relative oxidation and sheet-like growth
(Blackstone, 1999
).
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To further investigate such redox signaling, perturbations of colony growth and development were carried out using the hydroid Podocoryna carnea. Oxygen uptake of colonies was measured to determine comparable physiological doses of antimycin A1 (an inhibitor of complex III), rotenone (an inhibitor of complex I downstream of NADH dehydrogenase) and carbonyl cyanide m-chlorophenylhydrazone (CCCP; an uncoupler of oxidative phosphorylation). Genetically identical replicate colonies were grown at appropriate physiological doses, and colony development was measured. Using fluorescent microscopy of colonies treated with antimycin and rotenone, assays of peroxides were carried out with 2',7'-dichlorofluorescin diacetate. The data obtained from these experiments support the hypothesis that redox state and ROS are factors that mediate adaptive colony development, although alternative hypotheses are also discussed.
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Materials and methods |
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Measures of oxygen uptake
The actions of inhibitors and uncouplers can be gauged by the effects on
oxygen uptake; the former inhibit uptake, while the latter stimulate it.
Comparable physiological doses can thus be determined. For instance, if two
inhibitors at different concentrations produce the same effect on oxygen
uptake, a comparable physiological dose has been obtained. Similarly, if an
uncoupler at a particular concentration produces a similar, but inverse,
effect as these inhibitors, again a comparable physiological dose has been
obtained. Ideally, in all cases the perturbation of oxygen uptake should be
mild to moderate, i.e. physiologically appropriate, so as not to introduce any
pathological responses.
To determine comparable and appropriate physiological doses, standard
concentrations of inhibitors and uncouplers were used: 1 µmoll-1
CCCP, 1 µmoll-1 antimycin A1 and 10
µmoll-1 rotenone (e.g. Erecinska and Wilson, 1981;
Nishikawa et al., 2000). Stock
solutions of the inhibitors were prepared in ethanol, while the stock of CCCP
was prepared in seawater. For each compound, five assays were carried out;
five assays were also performed for each of the two controls (ethanol at the
appropriate concentration in seawater and plain seawater). For each of these
assays, a P. carnea colony on a 12 mm diameter cover slip was
attached with a drop of silicone grease to a cover slip cemented to a small
magnet. This assembly was contained in a 13 mm diameter sealed glass chamber
(RC300; Strathkelvin, Glasgow, UK) with 0.7 ml of seawater (filtered to 0.2
µm). Chamber temperature was held constant (20.5±0.02°C) using
an external circulation water bath (RTE-100D; Neslab, Newington, USA), and the
rate of decline in oxygen concentration over a 30 min period was measured
(using a Strathkelvin 1302 electrode and a 781 oxygen meter) with stirring (by
slowly spinning the magnet, cover slips and colony). The chamber was then
opened, a small volume of seawater removed, an equivalent amount of the
appropriate stock solution added to achieve the target concentration, the
solution mixed and aerated thoroughly with a small pipette, and the chamber
resealed (this procedure took approximately 7 min). The rate of decline in
oxygen concentration was then measured over another 30 min period. These
assays were performed 3-5 h after the feeding of the subject colony as part of
the normal feeding schedule. For each colony, the before/after difference in
the rate of decline in oxygen concentration over a 30 min period was
calculated, where this decline was measured by the least-squared slope of
oxygen concentration versus time. An overall trend in these
differences for the five colonies was analyzed using a paired-comparison
t-test. Furthermore, as the controls showed slight changes (the
ethanol control showed a slight decrease in oxygen consumption, while the
seawater control showed a slight increase), the change in slope for each
treatment was tested against the change in slope of the appropriate control
using a standard t-test.
Comparisons of colony growth and development
A series of experiments was carried out to investigate the effects of these
compounds on colony growth. Generally, the same target concentrations were
used as in the oxygen uptake experiments. Several preliminary experiments were
performed to determine the optimal time of exposure and to test the effects of
different solvents [ethanol and dimethyl sulfoxide (DMSO)]. Stocks of the
inhibitors and uncouplers were prepared at concentrations that allowed the
same solvent concentrations in all treatments. Controls were carried out at
these solvent concentrations (ethanol, 0.17%; DMSO, 0.08%). Although each of
these initial trials showed roughly similar trends (data not shown),
observations suggested that ethanol had a greater deleterious effect on the
colonies than DMSO. In part, this may be because the latter dissolved higher
stock concentrations of rotenone and thus allowed lower concentrations of
solvent in all treatments.
An in-depth follow-up experiment was thus performed using DMSO as the
solvent. Twenty-two replicates were explanted on 15 mm cover slips, with five
each assigned to control (0.08% DMSO) and to antimycin A1
treatments and six each assigned to rotenone and to CCCP treatments. Each
group was treated with the appropriate solution for approximately 4-6 h
day-1. As previously (e.g.
Blackstone, 1999), inhibitors
and uncouplers seemed to be best tolerated by colonies when treatment was
intermittent. As each colony initiated medusae production (up to 60 days after
explanting), that colony was imaged. Images were processed to facilitate
automatic measurement in Image-Pro Plus software (Mediacybernetics, Silver
Spring, USA). The gray level of some image objects (i.e. background, stolons
or polyps) was adjusted using Corel Photo-Paint software (background gray
level = 10, stolon = 201, polyp = 255). Processed images were checked against
the original images to ensure accuracy. Processed images were measured in
Image-Pro for the following parameters: total colony area and perimeter, total
cover slip area outside the colony, total unencrusted cover slip area within
the colony, and total polyp area.
Using PC-SAS software (SAS Institute, Cary, USA), the four treatments of this experiment were compared using univariate analysis of variance (ANOVA) and multivariate analysis of variance (MANOVA) for the relationship between the total area of polyps and the total area of empty, unencrusted cover slip both inside and outside the colony. Both polyp area and empty, unencrusted area were expressed as a fraction of the total surface area of the cover slip (176.71 mm2). To better meet the assumptions of parametric statistics, these data were arcsine transformed before analysis. Polyp area is clearly a measure of polyp development; empty, unencrusted cover slip area is a measure of how the colony covers and monopolizes space. This latter trait largely depends on stolon branching and anastomosis (i.e. as stolon development increases, empty cover slip area decreases). A second measure of stolon branching and anastomosis was also used: the mean size of the empty, unencrusted areas within the colony. These latter data were natural logarithm transformed before analysis with ANOVA.
Comparisons of ROS
Hydrogen peroxide represents a major component of ROS under physiological
conditions (Chance et al.,
1979), and measures of H2O2 were taken using
2',7'-dichlorofluorescin diacetate (H2DCFDA;
Jantzen et al., 1998
;
Nishikawa et al., 2000
;
Pei et al., 2000
). This
non-fluorescent dye is freely permeable to living cells. Once inside a cell,
H2DCFDA is deacetylated to H2DCF, which in turn
interacts with peroxides to form 2',7'-dichlorofluorescein, which
can then be visualized with fluorescent microscopy. The activation of
H2DCF is relatively specific for the detection of
H2O2 as well as secondary and tertiary peroxides.
Nevertheless, H2O2 is usually the major peroxide within
cells and is primarily measured by this method. A 10 mmol l-1 stock
solution of H2DCFDA was prepared in anhydrous DMSO. One day after
feeding, five colonies each were incubated in antimycin A1 and
rotenone (from stocks in DMSO) at the same concentrations as in the oxygen
uptake experiments. Within 1 h, H2DCFDA was added to a
concentration of 10 µmol l-1, and colonies were incubated for an
additional hour in the dark prior to measurement. Using a Hamamatsu Orca- 100
cooled-CCD camera and a Zeiss Axiovert 135, peroxide (as indicated by
H2DCFDA-derived 2',7'-dichlorofluorescein) was imaged
for an approximately 50 µmx150 µm region at the base of three
polyps per colony (excitation 450-490 nm, emission 515-565 nm). The major
metabolic signals from these colonies emanate from polyps, particularly the
epitheliomuscle cells near the junction of polyps and stolons. These cells
exhibit clusters of mitochondria surrounding their muscle fibers, and these
mitochondria provide strong metabolic signals relative to the remainder of the
colony (Blackstone, 1999
,
2001
). Images with 12-bit depth
(4096 gray levels) were thus obtained and were analyzed in Image-Pro. A major
conceptual difficulty in such an analysis is determining where to draw the
boundary between the foreground, bright areas (which constitute the area of
fluorescent signal) and the background (see for instance
Fig. 6 and
Blackstone, 2001
). This
consideration is particularly relevant here because H2O2
easily diffuses, and a concentration gradient is expected. Fortunately,
advances in image analysis technology allow an objective resolution of this
methodological difficulty. For each image, after the stolon area was outlined
as the region of interest, the software automatically identified the `bright
areas' (i.e. the area of signal) and the `dark areas' (i.e. the background),
using the same algorithm for each image. Total areas and average luminance of
both bright and dark areas were measured. Bright area luminance was corrected
for background luminance by subtraction, and the data were analyzed by a
nested ANOVA (polyp nested within replicate colony, replicate colony nested
within treatment).
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Results |
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Comparisons of colony growth and development
Each colony was imaged at the initiation of medusa production
(Fig. 3). A significant
treatment effect is apparent (MANOVA, F=9.3, d.f.=6, 34,
P<0.001), and this effect derives from an effect on both polyp
development (ANOVA, F=24.4, d.f.=3, 18, P<0.001) and
stolon development (ANOVA, F=15.6, d.f.=3, 18, P<0.001).
In essence, antimycin produces less polyp and stolon development and thus more
runner-like phenotypes than CCCP or rotenone. Strikingly, the most divergent
colony morphologies are produced by treatment with antimycin and rotenone
(Fig. 4).
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An analysis of the average size of the areas of empty cover slip within the colonies shows a similar pattern (Fig. 5). This measure reflects the branching and anastomosis of stolons within a colony, and there is a significant treatment effect (ANOVA, F=42.8, d.f.=3, 18, P<0.001). Indicative of more runner-like growth, colonies treated with antimycin have larger inner areas than the controls. Indicative of more sheet-like growth, colonies treated with CCCP and rotenone have smaller inner areas. Again, there is a clear difference between colonies treated with antimycin and rotenone, although in this case colonies treated with CCCP are slightly more divergent.
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Comparisons of reactive oxygen species
Peroxide, as indicated by H2DCFDA-derived
2',7'-dichlorofluorescein, provides a strong signal in the
epitheliomuscle cells at the base of polyps
(Fig. 6). At the fluorescein
excitation and emission wavelengths, negative controls show that there is
little native fluorescence under these conditions
(Blackstone, 2001).
Fluorescence at fluorescein wavelengths can thus be attributed to
H2DCFDA. There is a slight, but statistically significant,
difference between the fluorescence emitted by polyps treated with antimycin
and those treated with rotenone (Fig.
7; nested ANOVA, F=6.03, d.f.=1, 8,
P<0.05).
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Discussion |
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Interpreting these results depends critically on the observation that
direct feeding triggers high amplitude contractions of polyps
(Wagner et al., 1998;
Dudgeon et al., 1999
). In view
of these studies, the data consistently suggest that relative oxidation of
mitochondrial electron transport chains in polyp epitheliomuscular cells has a
permissive effect on polyp and stolon branch initiation. Relative oxidation
can be achieved either by the strong metabolic demand associated with
feeding-related contractions of these muscle cells or by treatment with
uncouplers of oxidative phosphorylation. On the other hand, relative reduction
of mitochondrial electron transport chains in polyps seems to have an
inhibitory effect on polyp and stolon branch initiation. Relative reduction
can be achieved either by the low metabolic demand associated with only
indirect feeding or by treatment with inhibitors of complexes III and IV.
Patterns of colony development thus correspond to distinct mitochondrial
redox states. Sheet-like growth corresponds to `state 3' (plentiful substrate
and high metabolic demand). Runner-like growth corresponds to `state 4'
(sufficient substrate and low metabolic demand;
Scheffler, 1999). Uncouplers
mimic state 3, while inhibitors of complexes III and IV mimic state 4. This
correlation suggests that signaling occurs between mitochondrial redox state
and pattern-forming genes located in the nucleus (cf.
Coffman and Davidson, 2001
).
Often, mitochondrial signaling is mediated by ROS
(Nishikawa et al., 2000
;
Brownlee, 2001
;
Echtay et al., 2002
;
Larsen and Clarke, 2002
;
Nemoto and Finkel, 2002
). The
correlation between redox state and peroxide levels in hydroid polyps is
suggestive in this regard (Blackstone,
2001
). More direct tests involve inhibiting electron transport at
specific complexes of the electron transport chain relative to sites of ROS
formation (Nishikawa et al., 2001). Here, rotenone was used to inhibit
electron transport downstream of the first site of ROS formation and upstream
of the second site (Fig. 1).
The resulting phenotypic effects were very similar to those produced by
uncouplers and strikingly different from those produced by antimycin or azide.
This suggests that signal transduction is initiated at or near the second site
of ROS generation. A similar pattern has also been found in other taxa, e.g.
bacteria (Georgellis et al.,
2001
) and mammals (Nishikawa et al., 2001). The latter work
implicates ROS in signal transduction, while the former implicates coenzyme Q.
In hydroids, a slight but statistically significant difference was found in
peroxide levels between polyps treated with rotenone and those treated with
antimycin. ROS may thus have a role in signal transduction in these organisms
as well.
Signaling from the second site of ROS generation may be more common than signaling from the first site because the former experiences greater electron flux. For instance, during typical metazoan aerobic metabolism, only electrons from NADH will pass through the first site. On the other hand, except for those few electrons donated to oxygen at the first site, electrons from both NADH and FADH2 will pass through the second site (Fig. 1). Factors that enhance the formation of FADH2 will exaggerate this effect. In particular, the relatively fat-rich diet of brine shrimp may explain why the second site of ROS formation is the principal locus of redox signaling in these hydroids. Acyl-CoA dehydrogenase, which catalyzes the first step in mitochondrial ß-oxidation of fatty acids, has an FAD cofactor and carries electrons to coenzyme Q, thus bypassing complex I. Further oxidation of fatty acids does produce NADH and ultimately acetyl-CoA, which is then metabolized in the tricarboxylic acid (TCA) cycle. The TCA cycle yields principally NADH, which of course carries electrons to the electron transport chain via complex I. Overall, such fatty acid metabolism probably produces considerably greater electron flux at the second site of ROS generation than at the first. This may explain the observed effects of rotenone as compared with antimycin.
Some consideration of these experimental results in a natural context is
perhaps warranted. Hydroids such as Podocoryna carnea usually encrust
gastropod shells inhabited by hermit crabs. Colony feeding may be affected by
various hermit crab-related factors (Van
Winkle et al., 2000), e.g. how the hermit crab holds the shell and
directs water currents over it. As hermit crabs molt, grow and switch shells
over a period of days and weeks, these factors probably vary. Food-related
perturbations of the growth of the hydroid colony would ensue, as generally
suggested in the Introduction. While the fat content of the hydroid diet in
nature is likely to be variable and, on average, less than that of brine
shrimp, a greater flux of electrons at the second site of ROS generation is
still expected (e.g. even with a carbohydrate diet). Thus, the second site
will consistently produce the bulk of the ROS, and the signaling pathway
suggested here will efficiently direct an adaptive response to feeding-related
perturbations.
The results presented here thus strongly support the hypothesis of redox signaling in the growth and development of these hydroid colonies. Furthermore, these results strongly implicate a signal emanating from between complexes I and III of the mitochondrial electron transport chain. This signal may consist of ROS, and there is some support for this hypothesis. Nevertheless, signaling via other intermediaries in addition to or instead of ROS cannot be ruled out at this time. Experiments that perturb the appropriate electron carriers and simultaneously block the effects of ROS are likely to be informative.
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Acknowledgments |
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