INTEGR. COMP. BIOL., 45:605–614 (2005)
Model Systems for Environmental Signaling1
NEIL W. BLACKSTONE2,*
AND
DIANE M. BRIDGE†
*Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois 60115
†Department of Biology, Elizabethtown College, One Alpha Drive, Elizabethtown, Pennsylvania 17022
SYNOPSIS. Studies of environmental signaling in animals have focused primarily on organisms with relatively constrained responses, both temporally and phenotypically. In this regard, existing model animals
(e.g., ‘‘worms and flies’’) are particularly extreme. Such animals have relatively little capacity to alter their
morphology in response to environmental signals. Hence, they exhibit little phenotypic plasticity. On the
other hand, basal metazoans exhibit relatively unconstrained responses to environmental signals and may
thus provide more general insight, insofar as these constraints are likely traits derived during animal evolution. Such enhanced phenotypic plasticity may result from greater sensitivity to environmental signals, or
greater abundance of suitable target cells, or both. Examination of what is known of the components of
environmental signaling pathways in cnidarians reveals many similarities to well-studied model animals. In
addition to these elements, however, macroscopic basal metazoans (e.g., sponges and cnidarians) typically
exhibit a system-level capability for integrating environmental information. In cnidarians, the gastrovascular
system acts in this fashion, generating local patterns of signaling (e.g., pressure, shear, and reactive oxygen
species) via its organism-wide functioning. Contractile regions of tissue containing concentrations of mitochondrion-rich, epitheliomuscular cells may be particularly important in this regard, serving in both a
functional and a signaling context. While the evolution of animal circulatory systems is usually considered
in terms of alleviating surface-to-volume constraints, such systems also have the advantage of enhancing the
capacity of larger organisms to respond quickly and efficiently to environmental signals. More general
features of animals that correlate with relatively unconstrained responses to environmental signals (e.g.,
active stem cells at all stages of the life cycle) are also enumerated and discussed.
INTRODUCTION: CONSTRAINED AND
UNCONSTRAINED PLASTICITY
In modern biology it is widely accepted that several
independent laboratories focusing on a particular
‘‘model organism’’ can make significant progress (e.g.,
Alberts et al., 2002). This approach has been repeatedly validated, as for instance by the Nobel Prize
award in 2002 for work on Caenorhabditis elegans
(Check, 2002, p. 549):
have a measurable morphological outcome. The nature
of the interaction between environmental signaling and
development can be best understood in animals that
differ strikingly from conventional model organisms
(Bolker, 1995).
In studies of development, the effect of environmental signaling on organismal phenotypes is usually
termed phenotypic plasticity. Considerable interest in
this topic is evident (Schlichting and Pigliucci, 1998;
Pigliucci, 2001; West-Eberhard, 2003). The role of
such environmental signaling is likely commensurate
with several organismal features related to longevity.
For instance, short-lived animals typically exhibit only
a brief window in which development will respond to
environmental signals, since the period of development itself is brief. Appropriate environmental signals
during this period may produce a response. The response itself is somewhat constrained, e.g., different
body or limb size and shape, but not more body axes.
Subsequently that response usually becomes fixed. The
organism may or may not survive and reproduce, but
typically no further effects of environmental signaling
will be seen. Such animals notably include rotifers
(Gilbert, 2003), crustaceans such as Daphnia (Tollrian
and Dodson, 1999), and many insects, including the
castes of social insects (Nijhout, 2003).
For instance, in the rotifer Asplanchna sieboldi, the
amount of dietary a-tocopherol (vitamin E, an antioxidant) determines the morphotype that will develop.
Morphotypes differ in body size and shape and type
of offspring produced (e.g., amictic versus mictic). Vitamin E exerts its primary influence on embryos when
they are developing in utero. While some effects can
Some scientists also see a larger significance to this
year’s Nobel, especially when combined with the
1995 Nobel for work in the fruitfly and the 2001
prize, which rewarded groundbreaking work in
yeast. ‘These awards are a recognition that you can
make major advances in medicine by studying genetically tractable model organisms,’ says Gerald
Rubin, a vice-president at the Howard Hughes Medical Institute in Chevy Chase, Maryland.
Nevertheless, it is unlikely that ‘‘worms and flies’’ are
sufficient for understanding animal diversity. Are there
general traits of animals that cannot be well studied
using these two model organisms? In this context, we
suggest that one such trait is environmental signaling,
here considered primarily in terms of developmental
pathways influenced by cues that initiate outside the
organism. In particular, we focus on pathways that
1 From the Symposium Model Systems for the Basal Metazoans:
Cnidarians, Ctenophores, and Placozoans presented at the Annual
Meeting of the Society for Integrative and Comparative Biology, 5–
9 January 2004, at New Orleans, Louisiana.
2 E-mail: [email protected]
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N. W. BLACKSTONE
be seen even at the late stages of embryogenesis, no
effects of tocopherol treatment are seen during postnatal development (Gilbert, 1980). Insects show similar kinds of plasticity, e.g., in butterfly polyphenisms
related to wing pattern, the environment-sensitive period occurs sometime during larval life (Nijhout,
2003). Subsequent to the metamorphic molt, no further
change occurs. In Daphnia, some phenotypic responses are irreversible (e.g., body size), while others can
be reversed over the course of several molts (e.g.,
neckteeth). The reversible traits are usually minor relative to the overall morphology and occur when the
within-generation variation in the environment is high
(Tollrian and Dodson, 1999).
Considering the physicochemical properties of developmental pathways, Nijhout (2003, p. 9) reasonably
suggests that ‘‘phenotypic plasticity is the primitive
character state for most if not all traits.’’ It follows that
restrictions or constraints on plasticity are derived features, possibly correlated with other derived features.
Animals such as rotifers, insects, and crustaceans that
usually have both temporally and phenotypically constrained responses to environmental signals typically
exhibit several interrelated features. Generally, reproduction occurs via a germ line. Asexual reproduction,
if present, is via amictic parthenogenesis. Fragmentation, budding, or other forms of agametic, asexual reproduction typically are not found. A more-or-less rigid cuticle often encases the animal. This cuticle may
constrain both environmental responses (Nijhout,
2003) and mode of reproduction (Blackstone and Jasker, 2003). A short life span is also typical. Notably,
the ecdysozoan clade that contains ‘‘worms and flies’’
generally exhibits all of these features (e.g., Fig. 1).
While some crustaceans are long-lived, at least as
adults these long-lived crustaceans likely inhabit relatively stable environments and also respond to environmental perturbations behaviorally (Tollrian and
Dodson, 1999).
On the other hand, consider organisms that reproduce by agametic, asexual reproduction. To a large extent, these organisms are continually patterning and repatterning such that they may respond to environmental signals continuously throughout their potentially
long life spans. A phenotypic effect can occur and be
maintained until the signal ceases, at which time the
normal pattern of development can be once again be
expressed. Further, phenotypic effects can be particularly dramatic, e.g., new body axes. Colonial animals
epitomize these generalities. Well-studied examples
occur in the hydractiniid hydroids, which encrust hard
substrata with stolons that serve as tube-like connections between feeding polyps. Upon contact with another such conspecific colony that does not share allorecognition alleles (see Cadavid, this volume) or a
colony of another species, a ‘‘rejection’’ response ensues (Buss et al., 1984; Lange et al., 1989). Stem cells
of the interstitial cell or I-cell lineage differentiate into
nematoblasts, which migrate to stolon tips and mature.
These stolon tips swell with nematocytes and project
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D. M. BRIDGE
FIG. 1. Character states of metazoans for agametic, asexual reproduction are mapped onto a phylogeny from Peterson and Eernisse
(2001). A filled box adjacent to a taxon indicates the presence of
agametic, asexual reproduction in at least one representative of that
taxon, while an unfilled box indicates the absence of this trait, at
least insofar as can be determined. Using parsimony, the filled nodes
are reconstructed as exhibiting agametic, asexual reproduction. Unfilled nodes indicate an absence of this trait (hatched nodes are ambiguous). Notably, the ecdysozoan clade containing nematodes and
insects has lost this trait (after Blackstone and Jasker, 2003).
above the substratum, discharging the nematocysts
into the neighboring colony (Fig. 2). This phenotype
may persist for months or longer until one colony is
killed (McFadden, 1986). Subsequently, the earlier
pattern of development resumes (Van Winkle and
Blackstone, 2002), although another encounter will
again provoke this ‘‘rejection’’ phenotype. Colonies
respond to other environmental signals in similar, if
less spectacular ways. Feeding a single polyp of a colony will result in concentrated growth of the colony
near the fed polyp and sparse growth elsewhere
(Blackstone, 2001). As detailed below, this effect is
mediated by the gastrovascular system of the colony
and by an abundant supply of stem cells. A number
of sometimes major developmental features of these
and other colonial organisms show similar patterns of
transient and recurring phenotypic plasticity (Harvell,
1999).
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FOR
ENVIRONMENTAL SIGNALING
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FIG. 2. Contact between a colony of Podocoryna carnea (top) and Hydractinia symbiolongicarpus (bottom). Hyperplastic stolons of the
former are visible in the area of contact, overlaying the stolonal mat of the latter (photo credit: D. H. Van Winkle).
The foregoing paragraphs introduce a simple theme:
the well-studied Caenorhabditis and Drosophila and
their relatives may share derived features that make
them relatively unresponsive to environmental signaling and thus less suitable for studying this phenomenon. Basal metazoans seem to lack these derived features. A number of other metazoan lineages may share
the plesiomorphic character states of basal taxa (e.g.,
Fig. 1). This unrestricted or unconstrained state of phenotypic plasticity likely involves continual activity of
complex environmental signaling pathways as well as
mitotically active target cells. Receptors detect the environmental stimuli, signals are transduced to the appropriate target cells, and the necessary second messenger systems activated, finally leading to transcription factors or pathways of post-translational protein
modification, or both. Such systems may be of particular interest in biology and medicine, e.g., in the human vascular system. The remainder of this paper further examines these ideas in three sections. First, we
outline the historical context, suggesting that developmental biology increasingly ignored environmental
effects as the field became more focused on Drosophila as an invertebrate model. Second, we highlight
what is known of environmental signaling pathways in
basal metazoans. We emphasize the well-studied cnidarians, but in principle these discussions may apply
to other early-evolving animals. The goal is to ascer-
tain what features allow these animals to be responsive
to environmental signals. Finally, given the patterns
elucidated in cnidarians and the themes touched on
above, we attempt to generalize: are there suites of
features that characterize constrained and unconstrained phenotypic plasticity?
HISTORICAL BACKGROUND: MODEL SYSTEMS MATTER
In the introduction to a recent Society for Integrative
and Comparative Biology symposium proceedings,
Gilbert and Bolker (2003) provide an excellent historical overview of why developmental biology has
largely ignored environmental effects (see also
Schlichting and Pigliucci, 1998). We complement
these and other historical treatments by emphasizing
the role of model organisms.
Early in the 20th century, a group of developmental
biologists did accord an important role to environmental signaling, particularly as it impacted metabolism.
As summarized by Bonner (1996), representative experiments were carried out on stalks of the hydroid
Tubularia. When severed from a colony, one end of
the stalk would regenerate a new hydranth. A series
of experiments demonstrated that if one end of the
stalk was buried in sand, the other end would always
regenerate the hydranth, regardless of the original polarity of the stalk. Advocates of environmental signaling suggested that gradients of oxygen diffusion
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caused this result, with the buried end receiving less
oxygen. The resulting oxygen gradient was hypothesized to affect metabolism and to lead to differential
development—the exposed end regenerated the hydranth.
In many ways, the publication of C. M. Child’s Patterns and Problems in Development (1941) marked the
zenith of theories linking such ‘‘metabolic activity’’
and related environmental signaling to organismal development. Subsequently, these theories were entirely
supplanted by the theory of variable gene activity, as
developed for instance by T. H. Morgan in Embryology
and Genetics (1934) and later by E. H. Davidson’s
Gene Activity in Early Development (1968). While
metabolic activity clearly subsumes aspects of gene
activity, Child and like-minded colleagues commonly
focused on energy metabolism and its manifestation
(e.g., oxygen uptake). Energy metabolism was thus
viewed as guiding gene activity, rather than the reverse. Given the sensitivity of aerobic energy metabolism to external inputs (e.g., substrate and oxygen
supply), the contrast to strict internal control was thus
sharp.
The demise of Child’s theories was at least in part
the result of social conflicts between groups of scientists who championed different model organisms (Mitman and Fausto-Sterling, 1992). Child and colleagues
(e.g., L. H. Hyman) worked primarily on hydroids and
planarians. As described above, these organisms contrast sharply to those that T. H. Morgan worked on—
fruit flies. Increased study of Drosophila led to diminished interest in Child’s chosen model organisms. For
instance, studies of planarians in the U.S. declined
sharply in the second half of the 20th century (Mitman
and Fausto-Sterling, 1992). The ascendancy of one
model system led to the subsequent decline of another.
Given the relatively constrained responses of Drosophila to environmental signals, a corresponding
view of development in general emerged.
From an historical perspective, the paper by Coffman and Davidson (2001) was a noteworthy milestone.
This paper provides evidence that the development of
the oral-aboral body axis in sea urchin embryos depends on gradients of oxygen concentration and their
influence on the metabolic state of the mitochondria.
While Davidson (1968) heralded the final demise of
Child’s theories, Coffman and Davidson (2001; see
also Coffman et al., 2004) indicate that at least some
of Child’s ideas continue to remain current even in the
21st century. It is noteworthy that deuterostomes (including sea urchins) may retain features of earlyevolving animals that are not retained by ecdysozoans
such as nematodes and insects (e.g., Fig. 1).
ENVIRONMENTAL SIGNALING PATHWAYS
IN
CNIDARIANS
Receptors
Cell level. Elaborate phenotypic responses can be
mediated in large part by cell-level receptors. The ‘‘rejection’’ response in hydractiniid hydroids serves as a
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general example. When an approaching stolon tip contacts another stolon, unidentified signals are apparently
emitted and received, and microbasic mastigophores
(nematocysts exclusive to the stolons) begin to accumulate near the area of contact (Lange et al., 1989).
These early-acting receptors are unrelated to histocompatibility, e.g., the same process occurs between stolons of the same colony. However, discharge of nematocysts only occurs after the neighboring stolon has
been identified as foreign, again likely by local celllevel receptors. Hyperplastic stolon formation occurs
from the accumulation of nematocytes in the stolonal
epidermis (Buss et al., 1984; Lange et al., 1989).
In members of the cnidarian genus Hydra, epithelial
tissue possesses antimicrobial activity (Kasahara and
Bosch, 2003). Antimicrobial activity differs in different body regions of the polyp, with lower activity in
tissue of the oral end, the region farthest from the substratum and substratum-associated bacteria (Kasahara
and Bosch, 2003). These results raise the possibility
of an inducible innate immune response in cnidarians.
System level. Aspects of the rejection response in
hydractiniid hydroids may suggest a system level response mediated by the gastrovascular system. For instance, discharge of nematocysts into a stolon will
temporarily halt gastrovascular flow (Lange et al.,
1989), and this may trigger other aspects of the response, for instance I-cell differentiation as well as
guiding nematocyte migration. The gastrovascular system may generally function in this fashion. Wyttenbach (1974) notes that ‘‘growth cycles’’ of stolon tips
are sensitive to a variety of environmental factors including temperature, sea water composition, nutritive
state, and feeding activity. At least some of these environmental effects may be mediated by responses of
the gastrovascular system. Buss (2001, p. 3) generalizes:
Clonal organisms typically posses one or more clonewide fluid-conducting systems (e.g., the phloem
system of vascular plants, the system of cytoplasmic
streaming within the hyphae of a fungal mycelium
or the plasmodium of a myxomycete, the gastrovascular system of cnidarians, and the blood vascular
system of certain colonial ascidians). The functioning of these systems will generate local patterns of
pressure distribution, shear stress, and surface tension experienced by the tissues, cells, and/or membranes lining the conducting system. If such features
can be detected and the signals transduced to effect
expression of pattern-forming genes, global rules responsive to local state arise.
Thus some environmental perturbations may be detected by components of the gastrovascular system,
which may then integrate the environmental inputs and
produce an altered pattern of system-level behavior. As
a result, the gastrovascular milieu of local signals that
cells experience shifts to an alternative state and a colony-wide response occurs, ultimately transduced by
local, cell-level receptors.
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FOR
ENVIRONMENTAL SIGNALING
609
FIG. 3. Photomicrograph of a colony of Podocoryna carnea stained with H2DCFDA (scale, 25 mm). Once acetate groups are removed by
intercellular esterases, H2DCF can be oxidized, typically by peroxides. Fluorescence indicates that this oxidation occurs primarily in the
mitochondrion-rich epitheliomuscular cells of polyp-stolon junctions (see Blackstone et al., 2004a, b). Stolons and polyps exhibit relatively
little fluorescence. Fluorescence of the chitinous perisarc is unrelated to H2DCF oxidation.
It remains a largely open question as to what components of the gastrovascular system detect and integrate environmental signals. At least in some hydroids,
regions of mitochondrion-rich epitheliomuscular cells
(EMCs) may have a central role in such colony-wide
responses (Blackstone et al., 2004a, b). Typically, hydroid EMCs show relatively low concentrations of mitochondria (e.g., Thomas and Edwards, 1991). Small
regions, perhaps consisting of dozens of EMCs, nevertheless exhibit an abundance of mitochondria. Depending on the species, these regions may be at polypstolon or polyp-stalk junctions, or alternatively forming a ring around the mouth. Contractions of these
mitochondrion-rich EMCs may in part drive or coordinate colony-wide gastrovascular flow. As well, these
mitochondria may emit signals correlated with redox
state, e.g., reactive oxygen species (ROS). Mitochondrial redox state is highly sensitive to environmental
inputs that alter electron sources and sinks (Allen,
1993). These regions of intense metabolic activity may
thus serve as integrators of environmental information
and ‘‘capacitors’’ of unconstrained development.
In hydractiniid hydroids, mitochondrion-rich EMCs
are found only at polyp-stolon junctions (Fig. 3), and
gastrovascular flow exhibits a characteristic response
to feeding (Dudgeon et al., 1999). Immediately following feeding, the polyp contracts but no food is released
into the stolons. After a short period of time, polyp
contractions result in the release of food-rich gastrovascular fluid into the stolons. The transition from contractions with no food release to contractions with food
release may depend on the behavior of the polyp-stolon junction, i.e., ‘‘closed’’ versus ‘‘open.’’ Subsequently, polyps and polyp-stolon junctions show coordinated activity, circulating food throughout the colony with a high rate of gastrovascular flow. The metabolic demand associated with these contractions
diminishes the EMC’s ATP/ADP ratio. In response,
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mitochondrial synthesis of ATP becomes maximal, and
the electrochemical gradient of the mitochondrial inner
membrane is thus diminished. Oxidation of substrate
and proton extrusion to rebuild this gradient is then
maximized, leaving the electron carriers of the electron
transport chain relatively oxidized. Oxidation of the
mitochondrial electron transport chain in turn diminishes the formation of ROS (Blackstone, 2001, 2003).
An increased gastrovascular flow thus directly affects
hydrodynamic signals such as pressure and shear
(Dudgeon and Buss, 1996) and indirectly affect redox
signals such as mitochondrial ROS (Blackstone, 2003;
Blackstone et al., 2004a, b). Local receptors for these
signals can lead to plastic development of the colony.
For instance, local feeding of one area of a colony
triggers greater polyp and stolon development in that
area (Blackstone, 2001).
Signal transduction
Shear stress. Generalizations about the movement
of fluids through branching systems imply that tissue
response to shear stress has the potential to produce
networks that allow energetically efficient flow. Murray’s Law describes a network architecture that minimizes the energetic cost of moving fluid through a
network of branching vessels. In networks conforming
to Murray’s Law, shear stress on the walls of all vessels in equal. Thus, a system of vessels following Murray’s law could be produced if changes in vessel diameter and/or new vessel formation were responsive
to shear stress (for review, see LaBarbera, 1990). In
the vertebrate vascular system, shear stress exerted by
blood flow has substantial effects on endothelial cell
morphology and behavior. Molecules potentially involved in sensing shear stress in endothelial cells include integrins and other focal adhesion components
(Jalali et al., 2001; Tzima et al., 2001), vascular endothelial growth factor receptor 2 (Shay-Salit et al.,
2002), platelet endothelial cell adhesion molecule-1
(Osawa et al., 2002), and ion channels (Brakemeier et
al., 2002; Hoger et al., 2002). Shear stress triggers
signaling through multiple pathways. Responses to
shear stress include activation of mitogen-activated
protein (MAP) kinases (Li et al., 1997), Src family
kinases (Jalali et al., 1998), and G proteins (Gudi et
al., 1996), and elevated levels of inositol triphosphate
and diacylglycerol (DAG) (Nollert et al., 1990).
Among the effects of shear stress-induced signaling
are changes in the synthesis of the vasoconstrictor endothelin-1 (Sharefkin et al., 1991).
A number of the molecules involved in responses
of vertebrate cells to shear stress have been studied in
cnidarians. Integrin genes have been identified in both
the coral Acropora millepora (Brower et al., 1997) and
the hydractiniid hydroid Podocoryna carnea (ReberMuller et al., 2001). A Src family gene has been
cloned from the solitary hydrozoan Hydra vulgaris
(Bosch et al., 1989). DAG has been shown to affect
tissue fate in Hydra and the hydractiniid hydroid Hydractinia echinata, inducing formation of secondary
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polyps on the body column (Muller, 1989). DAG is an
activator of protein kinase C; two protein kinase C
genes have been isolated from Hydra vulgaris (Hassel,
1998; Hassel et al., 1998). Finally, an endothelin-converting enzyme gene has been cloned from Hydra vulgaris, and human endothelin-1 has been shown to induce muscular contraction in Hydra polyps (Zhang et
al., 2001).
Reactive oxygen species. Production of ROS by mitochondria may influence or be influenced by signal
transduction in any or all of several ways. (1) Nitric
oxide (NO) can act as an inter- and intracellular messenger. Originally identified as a vasodilator, NO also
interacts extensively with mitochondria, e.g., at high
levels inhibiting cytochrome oxidase, while at low levels upregulating PGC-1a, a transcriptional coactivator
that regulates mitochondrial biogenesis (e.g., Brown,
2003; Leary and Shoubridge, 2003). In turn, ROS produced by mitochondria can interact with nitric oxide
and other reactive nitrogen species in a variety of ways
(Levonen et al., 2001). (2) In animal cells, mitochondria integrate various internal signals and, in response,
may release factors such as cytochrome c that activate
caspases and initiate programmed cell death. Oxidative
stress is one of the general internal signals that affect
mitochondria, and ROS in turn contribute to levels of
oxidative stress (Duan et al., 1999; Ueda et al., 2002).
Mitochondria have a prominent role in cnidarian apoptosis (see David et al., 2005). (3) In its reduced
state, the amino acid cysteine is a thiol; when oxidized
it can form disulfide bonds or other structures. Proteins
containing cysteine can thus have one conformation
when reduced and another when oxidized. Either or
both forms of the protein may be functionally important. While thioredoxins or other enzymes often catalyze the formation of disulfide bonds (e.g., Kadokura
et al., 2004), ROS, particularly peroxide, contribute to
an oxidizing environment that favors such reactions.
A number of proteins have their activity regulated in
this way (e.g., tyrosine phosphatases; Salmeen et al.,
2003; van Montfort et al., 2003). Putative homologs
of many of these proteins can be found in H. magnipapillata ESTs (see Steele, this volume). (4) Proteins
with iron-sulfur clusters in enzymatically active forms
can be inactivated by ROS, particularly superoxide.
For instance, the mitochondrial enzyme aconitase,
which converts citrate to isocitrate thus launching the
citric acid cycle, is reversibly inactivated by superoxide (Walden, 2002).
Vertebrate endothelial cells exhibit substantial crosstalk between signaling associated with shear stress and
signaling associated with reactive oxygen species. For
example, shear stress leads to NO release (Cooke et
al., 1991), and ROS production is required for shear
stress-induced MAP kinase activation (Yeh et al.,
1999). Thus, in the vertebrate vascular system, signals
from flow-related shear stress and from tissue redox
state are integrated. We might expect this to be the
case in basal metazoans as well, but its importance
remains to be determined.
MODEL SYSTEMS
FOR
ENVIRONMENTAL SIGNALING
Post-translational protein modification
Reactive oxygen species. As described above, ROS
can affect post-translational modification of proteins in
several ways. Reversible oxidation of a cysteine residue often occurs. For example, protein tyrosine phosphatases contain a catalytic cysteine (van Montfort et
al., 2003). When this cysteine is oxidized, usually by
H2O2, tyrosine phosphatases are inhibited. Target protein dephosphorylation is suppressed, and signal transduction pathways mediated by tyrosine kinases are optimally activated (Salmeen et al., 2003). On the other
hand, peroxiredoxins directly mediate H2O2 signaling
via their antioxidant function. When H2O2 is reduced,
the catalytic cysteine residue is oxidized. At low concentrations of peroxide, a disulphide bond will form
with the cysteine from the other subunit of the homodimer. Thioredoxin subsequently reduces the disulfide bond. However, at high concentrations of peroxide, the catalytic cysteine may react with H2O2 again
to form higher oxidation products, which cannot be
reduced by thioredoxin. Such overoxidation of peroxiredoxins then leaves peroxide available to participate in other signaling functions (Georgiou and Masip,
2003). In zinc finger proteins such as SAG, at least
two cysteines are necessary to form the zinc finger
structure. The thiol/disulfide equilibrium interacts with
the properties of zinc to produce potentially sophisticated redox behavior (Duan et al., 1999; Lee and Maret, 2001). Finally, proteins with iron-sulfur clusters in
enzymatically active forms can be inactivated by ROS,
usually reversibly (Walden, 2002). Superoxide, not
peroxide, interacts with these iron-sulfur clusters.
These various redox-mediated protein modifications
seem to be a shared feature of eukaryotes, and EST
data from H. magnipapillata (see Steele, 2005) indicate that cnidarians are not exceptions to this general
rule (Blackstone et al., 2004b).
DISCUSSION: ARE THERE GENERAL FEATURES
UNCONSTRAINED PLASTICITY?
OF
Botanists pioneered studies of phenotypic plasticity
(e.g., Schlichting and Pigliucci, 1998), and plants continue to be widely studied in this context (Sultan,
2003). As Buss (2001) points out, fungi are also appropriate for such studies. Nevertheless, if a goal of
studying phenotypic plasticity is to provide insight into
animals in general and perhaps human beings in particular, then animal models of phenotypic plasticity
must be developed. It is thus perhaps surprising that
studies of plasticity in animals have focused on taxa
that exhibit relatively constrained responses, both temporally and phenotypically. Such constraints were likely derived in the course of animal evolution (Nijhout,
2003). Of course, basal taxa may themselves exhibit
derived character states (e.g., see Collins et al., 2005).
However, sponges, cnidarians, and placozoans as well
as fungi and plants all exhibit relatively unconstrained
responses to environmental signals. The consensus of
character states among these three basal metazoan taxa
611
as well as relevant outgroups supports the hypothesis
that this is the ancestral metazoan character state. With
the exception of ctenophores, basal metazoans may
thus serve as useful model systems for the study of
environmental signaling and phenotypic plasticity.
Such model systems for environmental signaling may
better illuminate this phenomenon in animals in general.
While general patterns of ‘‘system-level’’ regulation
may be common to plants, fungi, and colonial animals,
many of the pathways of signal transduction governed
by these systems can be expected to be different. Proteins important in cell-cell signaling in animals, including tyrosine kinases, cadherins, and C-type lectins,
appear to be present in choanoflagellates, a probable
outgroup to animals, but not in plants or fungi (King
et al., 2003). Components of diverse animal signaling
pathways that have not been identified in plants or
fungi have been found in cnidarians (reviewed in Darling et al., 2005; Steele, 2002). On the other hand,
although animals may share the basic cell and molecular components of signaling, animal responses to environmental signals nonetheless differ dramatically.
Consideration of the basal metazoans may illuminate these differences. For instance, sponges and colonial cnidarians in particular show interesting parallels in body organization. Some members of both phyla have a modular organization with feeding, fluidmoving structures or regions (e.g., choanocyte
chambers or contractile polyp-stolon junctions), connected by presumably less metabolically active fluidbearing canals. Given these parallels in organization,
shear stress and ROS-mediated signaling could similarly orchestrate the spacing of metabolically active
regions in sponges and colonial cnidarians. A study of
vessel networks in fossil sponges provides indirect evidence for regulation of network development by shear
stress, since vessel diameters in the sponges studied
conform to Murray’s Law (LaBarbera, 1990). SSU
rDNA data suggest that sponges may be paraphyletic
(Borchiellini et al., 2001; Medina et al., 2001; Peterson and Eernisse, 2001). This raises the interesting
possibility that the organization characterizing sponges
and many cnidarians could be a shared primitive feature of the Metazoa. Further work on signaling in
sponges and colonial cnidarians should help to resolve
this issue. Growth and remodeling of vertebrate vascular systems are responsive to both shear stress and
ROS (reviewed in Davies et al., 2003; Pugh and Ratcliffe, 2003). Information from basal metazoans will
allow us to determine whether mechanisms regulating
the human vascular system’s organization indeed date
back to early colonial animals.
In basal metazoans and in animals in general, the
capacity for phenotypic plasticity may correlate with
other features, and we consider the evolution of these
and other features related to animal development. We
propose (Table 1) that animals that lack a sequestered
germ line but exhibit agametic, asexual reproduction,
active stem cells at all stages of the life cycle, and
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TABLE 1. Features of organisms with relatively unconstrained responses to environmental signals (and hence extensive phenotypic
plasticity) compared to those found in organisms with relatively constrained responses and hence limited phenotypic plasticity.
Trait
Germ line
Unconstrained
absent or sequestered
late
Asexual reproduction
usually agametic
Somatic stem cells
continually active
Longevity
potentially long
Environmental signaling continuous
System-level signaling
pervasive
Examples
sponges, cnidarians
Contrained
sequestered early
usually gametic
limited activity
usually short
episodic
circumscribed
insects, nematodes
potentially long life spans will also exhibit relatively
unconstrained phenotypic plasticity. Both successful
agametic, asexual reproduction and successful longevity likely require environmentally sensitive signaling
pathways. For instance, in Hydra, budding rates are
highly sensitive to the abundance of food, and poorly
fed specimens fail to bud (see David et al., 2005). In
the sea anemones Metridium senile and Aiptasia californica, increased movement of the substratum causes
an increased rate of asexual reproduction through pedal laceration (Anthony and Svane, 1995; Geller et al.,
2005). Similarly, environmentally sensitive ‘‘remodeling’’ within an organism can successfully prolong
life span in response to environmental insults, as is
typically seen in the human vascular system. At the
same time, the related traits of the lack of or late sequestration of a germ line and presence of mitotically
active stem cells provide the targets for environmental
signaling pathways to act on. For example, in the ‘‘rejection’’ response in colonical hydractiniid hydroids,
stem cells differentiate to nematocytes, which migrate
to stolon tips and produce the characteristic phenotype
of the hyperplastic stolons.
Long-lived animals capable of agametic, asexual reproduction and exhibiting mitotically active stem cells
are most likely to exhibit pathways of environmental
signaling that are continually active and that may involve relatively large-scale phenotypic responses. Except in microscopic animals such as placozoans, an
efficient way to do this is via ‘‘system level’’ regulation of signaling pathways, typically using some sort
of circulatory or vascular system. While such circulatory systems are generally regarded as a solution to
surface-to-volume constraints, an important related
function may be to allow larger organisms to respond
quickly and efficiently to environmental perturbation.
Circulatory systems essentially bring environmental
signals to all cells and tissues of a large multicellular
organism. The evolution of such systems must be considered in the context of signaling as well as in the
context of increasing surface relative to volume. Put
another way, increasing surface relative to volume enhances the reception of environmental signals. The
evolutionary constraints on circulatory systems will
furthermore depend on cellular responses to environmental signals. For instance, if the cellular response to
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D. M. BRIDGE
hypoxia is a robust anaerobic metabolism, an animal’s
circulatory system will function under a very different
set of constraints than if the cellular response to hypoxia is apoptosis.
Basal metazoans have elicited considerable interest
as sources of information about the evolution of animal embryonic development. We suggest that these
early-evolving animals may provide similar insight
into environmental signaling and phenotypic plasticity.
While all animals may share basic cellular and molecular components of environmental signaling pathways,
the responses generated by these pathways may nevertheless differ. Early-evolving animals seem to have
relatively unconstrained responses to environmental
signals, in contrast to at least some of the more derived
animals (e.g., ‘‘worms and flies’’). In this context, further studies of the former are certainly warranted.
ACKNOWLEDGMENTS
Many thanks to the Society for Integrative and
Comparative Biology in general and the Divisions of
Invertebrate Zoology and Evolutionary Developmental
Biology in particular for hosting and supporting this
symposium. The National Institutes of Health, the National Science Foundation, and Northern Illinois University also provided generous support. Helpful comments on this manuscript were provided by Molly Rorick, by four anonymous reviewers, and by the editor,
Alan Kohn.
REFERENCES
Alberts, B., A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter.
2002. Molecular biology of the cell. 4th ed. Garland Science,
New York.
Allcn, J. F. 1993. Control of gene expression by rcdox potential and
the requirement for chloroplast and mitochondrial genomes. J.
Theor. Biol. 165:609–631.
Anthony, K. R. N. and I. Svane. 1995. Effects of substratum instability on locomotion and pedal laccration in Metridium sentile
(Anthozoa: Actinaria). Mar. Ecol. Prog. Ser. 124:171–180.
Blackstone, N. W. 2001. Redox state, reactive oxygen species, and
adaptive growth in colonial hydroids. J. Exp. Biol. 204:1845–
1853.
Blackstone, N. W. 2003. Redox signaling in the growth and development of colonial hydroids. J. Exp. Biol. 206:651–658.
Blackstone, N. W., K. S. Cherry, and S. L. Glockling. 2004a. Structure and signaling in polyps of a colonial hydroid. Invert. Biol.
123:43–53.
Blackstone, N. W., K. S. Cherry, and D. H. Van Winkle. 2004b. The
role of polyp-stolon junctions in the redox signaling of colonial
hydroids. Hydrobiologia 530/531:291–298.
Blackstone, N. W. and B. D. Jasker. 2003. Phylogenetic considerations of clonality, coloniality, and mode of germline development in animals. J. Exp. Zool. (MDE) 297B:35–47.
Bolker, J. A. 1995. Model systems in developmental biology.
BioEssays 17:451–455.
Bonner, J. T. 1996. Sixty years of biology. Princeton University
Press, Princeton, New Jersey.
Borchiellini, C., M. Manuel, E. Alivon, N. Boury-Esnault, J. Vacelet, and Y. Le Parco. 2001. Sponge paraphyly and the origin of
Metazoa. J. Evol. Biol. 14:171–179.
Bosch, T. C. G., T. F. Unger, D. A. Fisher, and R. E. Steele. 1989.
Structure and expression of STK, a src-related gene in the simple metazoan Hydra attenuata. Molec. Cell Biol. 9:4141–4151.
Brakemeier, S., I. Eichler, H. Hopp, R. Kohler, and J. Hoyer. 2002.
MODEL SYSTEMS
FOR
ENVIRONMENTAL SIGNALING
Up-regulation of endothelial stretch-activated cation channels
by fluid shear stress. Cardiovasc. Res. 53:209–218.
Brower, D. L., S. M. Brower, D. C. Hayward, and E. E. Ball. 1997.
Molecular evolution of integrins: Genes encoding integrin beta
subunits from a coral and a sponge. Proc. Natl. Acad. Sci.
U.S.A. 94:9182–7.
Brown, G. C. 2003. NO says yes to mitochondria. Science 299:838–
839.
Buss, L. W. 2001. Growth by intussusception in hydractiniid hydroids. In J. B. C. Jackson, S. Lidgard, and F. K. McKinney
(eds.), Evolutionary patterns, pp. 3–26. University of Chicago
Press, Chicago, Illinois.
Buss, L. W., C. McFadden, and D. R. Keene. 1984. Biology of
hydractiniid hydroids. 2. Histocompatibility effector system/
competitive mechanism mediated by nematocyst discharge.
Biol. Bull. 167:139–158.
Cadavid, L. F. 2005. Self/non-self discrimination in basal metazoans:
Genetics of allorecognition in the hydroid Hydractinia symbiolongicarpus. Integr. Comp. Biol. 45:623–630.
Child, C. M. 1941. Patterns and problems in development. University of Chicago Press, Chicago, Illinois.
Check, E. 2002. Worm cast in starring role for Nobel Prize. Nature
419:548–549.
Coffman, J. A. and E. H. Davidson. 2001. Oral-aboral axis specification in the sea urchin embryo. Dev. Biol. 230:18–28.
Coffman, J. A., J. J. McCarthy, C. Dickey-Sims, A. J. Robertson.
2004. Oral-aboral axis specification in the sea urchin embryo
II. Mitochondrial distribution and redox state contribute to establishing polarity in Strongylocentrotus purpuratus. Dev. Biol.
273:160–171.
Collins, A. G., P. Cartwright, C. S. McFadden, and B. Schierwater.
2005. Phylogenetic context and basal metazoan model systems.
Integr. Comp. Biol. 45:585–594.
Cooke, J. P., E. Rossitch, N. A. Andon, J. Loscalzo, and V. J. Dzau.
1991. Flow activates an endothelial potassium channel to release an endogenous nitrovasodilator. J. Clin. Invest. 88:1663–
1671.
Darling, J. A., A. R. Reitzel, P. M. Burton, M. E. Mazza, J. F. Ryan,
J. C. Sullivan, and J. R. Finnerty. 2005. Rising starlet: the starlet
sea anemone Nematostella vectensis. Bioessays 27:211–221.
David, C. N., N. Schmidt, M. Schade, B. Pauly, O. Alexandrova,
and A. Böttger, 2005. Hydra and the evolution of apoptosis.
Integr. Comp. Biol. 45:631–638.
Davidson, E. H. 1968. Gene activity in early development. Academic
Press, New York.
Davies, P. F., J. Zilberberg, and B. P. Helmke. 2003. Spatial microstimuli in endothelial mechanosignaling. Cir. Res. 92:359–370.
Duan, H., Y. Wang, M. Aviram, M. Swaroop, J. A. Loo, J. Bian, Y.
Tian, T. Mueller, C. L. Bisgaier, and Y. Sun. 1999. SAG, a novel
zinc RING finger protein that protects cells from apoptosis induced by redox agents. Mol. Cell. Biol. 19:3145–3155.
Dudgeon, S. R. and L. W. Buss. 1996. Growing with the flow: On
the maintenance and malleability of colony form in the hydroid
Hydractinia. Am. Nat. 147:667–691.
Dudgeon, S. R., A. Wagner, J. R. Vaisnys, and L. W. Buss. 1999.
Dynamics of gastrovascular circulation in the hydrozoan Podocoryne carnea: The one-polyp case. Biol. Bull. 196:1–17.
Geller, J. B., L. J. Fitzgerald, and C. E. King. 2005. Fission in sea
anemones: Integrative studies of life cycle evolution. Integr.
Comp. Biol. 45:615–622.
Georgiou, G. and L. Masip. 2003. An overoxidation journey with a
return ticket. Science 300:592–594.
Gilbert, J. J. 1980. Developmental polymorphism in the rotifer Asplanchna sieboldi. Amer. Sci. 68:636–646.
Gilbert, J. J. 2003. Environmental and endogenous control of sexuality in a rotifer life cycle: Development and population biology. Evol. Dev. 5:19–24.
Gilbert, S. F. and J. A. Bolker. 2003. Ecological developmental biology: Preface to the symposium. Evol. Dev. 5:3–8.
Gudi, S. R. P., C. B. Clark, and J. A. Frangos. 1996. Fluid flow
rapidly activates G proteins in human endothelial cells: Involvement of G proteins in mechanochemical signal transduction.
Circ. Res. 79:834–839.
613
Harvell, C. D. 1999. Complex biotic environments, coloniality, and
heritable variation for inducible defenses. In R. Tollrian and C.
D. Harvell (eds.), The ecology and evolution of inducible defenses, pp. 231–244. Princeton University Press, Princeton, New
Jersey.
Hassel, M. 1998. Upregulation of a Hydra vulgaris cPKC gene is
tightly coupled to the differentiation of head structures. Dev.
Genes Evol. 207:489–501.
Hassel, M., D. M. Bridge, N. A. Stover, H. Kleinholz, and R. E.
Steele. 1998. The level of expression of a protein kinase C gene
may be an important component of the patterning process in
Hydra. Dev. Genes Evol. 207:502–14.
Hoger, J. H., V. I. Ilyin, S. Forsyth, and A. Hoger. 2002. Shear stress
regulates the endothelial Kir2.1 ion channel. Proc. Natl. Acad.
Sci. U.S.A. 99:7780–7785.
Jalali, S., Y. S. Li, M. Sotoudeh, S. Yuan, S. Li, S. Chien, and J. Y.
Shyy. 1998. Shear stress activates p60src-Ras-MAPK signaling
pathways in vascular endothelial cells. Arterioscler. Thromb.
Vasc. Biol. 18:227–34.
Jalali, S., M. A. del Pozo, K. D. Chen, H. Miao, Y. S. Li, M. A.
Schwartz, J. Y. J. Shyy, and S. Chien. 2001. Integrin-mediated
mechanotransduction requires its dynamic interaction with specific extracellular matrix (EMC) ligands. Proc. Natl. Acad. Sci.
U.S.A. 98:1042–1046.
Kadokura, H., H. Tian, T. Zander, J. C. A. Bardwell, and J. Beckwith. 2004. Snapshots of DsbA in action: Detection of proteins
in the process of oxidative folding. Science 303:534–537.
Kasahara, S. and T. C. G. Bosch. 2003. Enhanced antibacterial activity in Hydra polyps lacking nerve cells. Dev. Comp. Immunol. 27:79–85.
King, N., C. T. Hittinger, and S. B. Carroll. 2003. Evolution of key
cell signaling and adhesion protein families predates animal origins. Science 301:361–363.
LaBarbera, M. 1990. Principles of design of fluid transport systems
in zoology. Science 249:992–1000.
Lange, R., G. Plickert, and W. A. Müller. 1989. Histoincompatibility
in a low invertebrate, Hydractinia echinata: Analysis of a mechanism of rejection. J. Exp. Zool. 249:284–292.
Leary, S. C. and E. A. Shoubridge. 2003. Mitochondrial biogenesis:
Which part of ‘‘NO’’ do we understand? BioEssays 25:538–
541.
Lee, S.-H. and W. Maret. 2001. Redox control of zinc finger proteins: Mechanisms and role in gene regulation. Antiox. Redox
Signal. 3:531–534.
Levonen, A.-L., R. P. Patel, P. Brookes, Y.-M. Go, H. Jo, S. Parthasarathy, P. G. Anderson, and V. M. Darley-Usmar. 2001.
Mechanisms of cell signaling by nitric oxide and peroxynitrite:
From mitochondria to MAP kinases. Antiox. Redox Signal. 3:
215–229.
Li, S., M. Kim, Y. L. Hu, S. Jalai, D. D. Schlaepfer, T. Hunter, S.
Chien, and J. Y. Shyy. 1997. Fluid shear stress activation of
focal adhesion kinase. Linking to mitogen-activated protein kinases. J. Biol. Chem. 272:30455–30462.
McFadden, C. S. 1986. Laboratory evidence for a size refuge in
competitive interactions between the hydroids Hydractinia
echinata (Flemming) and Podocoryne carnea (Sars). Biol. Bull.
171:161–174.
Medina, M., A. G. Collins, J. D. Silberman, and M. L. Sogin. 2001.
Evaluating hypotheses of basal animal phylogeny using complete sequences of large and small subunit rRNA. Proc. Natl.
Acad. Sci. U.S.A. 98:9707–12.
Mitman, G. and A. Fausto-Sterling. 1992. Whatever happened to
Planaria? C. M. Child and the physiology of inheritance. In A.
E. Clarke and J. H. Fujimura (eds.), The right tools for the job,
pp. 172–197. Princeton University Press, Princeton, New Jersey.
Morgan, T. H. 1934. Embryology and genetics. Columbia University
Press, New York.
Müller, W. A. 1989. Diacylglycerol-induced multihead formation in
Hydra. Development 105:306–316.
Nijhout, H. F. 2003. Development and evolution of adaptive polyphenisms. Evol. Dev. 5:9–18.
Nollert, M. U., S. G. Eskin, and L. V. McIntire. 1990. Shear stress
614
N. W. BLACKSTONE
increases inositol trisphosphate levels in human endothelial
cells. Biochem. Biophys. Res. Commun. 170:281–287.
Osawa, M., M. Masuda, K. Kusano, and K. Fujiwara. 2002. Evidence for a role of PECAM-1 in endothelial cell mechanosignal
transduction: Is it a mechanoresponsive molecule? J. Cell. Biol.
158:773–785.
Peterson, K. J. and D. J. Eernisse. 2001. Animal phylogeny and the
ancestry of bilaterians: Inferences from morphology and 18S
rDNA gene sequences. Evol. Dev. 3:170–205.
Pigliucci, M. 2001. Phenotypic plasticity: Beyond nature and nurture. Johns Hopkins University Press, Baltimore, Maryland.
Pugh, C. W. and P. J. Ratcliffe. 2003. Regulation of angiogenesis by
hypoxia: Role of the HIF system. Nat. Med. 9:677–684.
Reber-Muller, S., R. Studer, P. Muller, N. Yanze, and V. Schmid.
2001. Integrin and talin in the jellyfish Podocoryne carnea. Cell
Biol. Int. 25:753–769.
Salmeen, A., J. N. Andersen, M. P. Meyers, T.-C. Meng, J. A. Hinks,
N. K. Tonks, and D. Barford. 2003. Redox regulation of protein
tyrosine phosphatase 1B involves a suphenyl-amide intermediate. Nature 423:769–773.
Schlichting, C. D. and M. Pigliucci. 1998. Phenotypic evolution.
Sinauer, Sunderland, Massachusetts.
Sharefkin, J. B., S. L. Diamond, S. G. Eskin, L. V. McIntire, and C.
W. Dieffenbach. 1991. Fluid flow decreases preproendothelin
mRNA levels and suppresses endothelin-1 peptide release in
cultured human endothelial cells. J. Vasc. Surg. 14:1–9.
Shay-Salit, A., M. Shushy, E. Wolfovitz, H. Yahav, F. Breviario, E.
Dejana and N. Resnick. 2002. VEGF receptor 2 and the adherens junction as a mechanical transducer in vascular endothelial
cells. Proc. Natl. Acad. Sci. U.S.A. 99:9462–9467.
Steele, R. E. 2002. Developmental signaling in Hydra: What does
it take to build a ‘‘simple’’ animal? Dev. Biol. 248:199–219.
Steele, R. E. 2005. Genomes of basal metazoans. Integr. Comp. Biol.
45:639–648.
Sultan, S. E. 2003. Phenotypic plasticity in plants: A case study in
ecological development. Evol. Dev. 5:25–33.
AND
D. M. BRIDGE
Thomas, M. B. and N. C. Edwards. 1991. Cnidaria: Hydrozoa. In F.
W. Harrison and J. A. Westfall (eds.), Microscopic anatomy of
the invertebrates, Vol. 2, pp. 91–183. Wiley-Liss, New York.
Tollrian, R. and S. I. Dodson. 1999. Inducible defenses in cladocera:
Constraints, costs, and multipredator environments. In R. Tollrian and C. D. Harvell (eds.), The ecology and evolution of
inducible defenses, pp. 177–202. Princeton University Press,
Princeton, New Jersey.
Tzima, E., M. A. del Pozo, S. J., Shattil, S. Chien, and M. A.
Schwartz. 2001. Activation of integrins in endothelial cells by
fluid shear stress mediated Rho-dependent cytoskeletal alignment. EMBO 20:4639–4647.
Ueda, S., H. Masutani, H. Nakamura, T. Tanaka, M. Ueno, and J.
Yodoi. 2002. Redox control of cell death. Antiox. Redox Signal.
4:405–414.
van Montfort, R. L. M., M. Congreve, D. Tisi, R. Carr, and H. Jhoti.
2003. Oxidation state of the active-site cysteine in protein tyrosine phosphatase 1B. Nature 423:773–777.
Van Winkle, D. H. and N. W. Blackstone. 2002. Variation in growth
and competitive ability between sexually and clonally produced
hydroids. Biol. Bull. 202:156–165.
Walden, W. E. 2002. From bacteria to mitochondria: Aconitase
yields surprises. Proc. Natl. Acad. Sci. U.S.A. 99:4138–4140.
West-Eberhard, M. J. 2003. Developmental plasticity and evolution.
Oxford University Press, Oxford, UK.
Wyttenbach, C. R. 1974. Cell movements associated with terminal
growth in colonial hydroids. Amer. Zool. 14:699–717.
Yeh, L.-H., Y. J. Park, R. J. Hansalia, I. S. Ahmed, S. S. Deshpande,
P. J. Goldschmidt-Clermont, K. Irani, and R. B. Alevriadou.
1999. Shear-induced tyrosine phosphorylation in endothelial
cells requires Rac1-dependent production of ROS. Am. J. Physiol. 276:C838–47.
Zhang, J., A. Leontovich, and M. P. Sarras, Jr. 2001. Molecular and
functional evidence for early divergence of an endothelin-like
system during metazoan evolution: Analysis of the Cnidarian,
hydra. Development. 128:1607–15.