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Mitochondria and the redox control of development in cnidarians
Seminars in Cell & Developmental Biology 20 (2009) 330–336
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Review
Mitochondria and the redox control of development in cnidarians Neil Blackstone ∗ Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115, USA
a r t i c l e
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Article history: Available online 24 December 2008 Keywords: Coral bleaching Cnidarian Mitochondrion-rich cell Reactive oxygen species Redox signaling
a b s t r a c t Mitochondria are the product of an ancient symbiosis between bacteria and host cells. While mitochondria function primarily in energy conversion, increasing amounts of evidence indicate that mitochondrial metabolic state can influence various emergent features of eukaryotic cells. Important intermediaries in such redox signaling include by-products of metabolism, particularly reactive oxygen species (ROS). This review uses cnidarians, a group of basally branching animals, to illustrate the many and varied effects of ROS on development. ROS from both mitochondria and algal symbionts are considered. Because some applications of ROS may lack specificity, the signaling demands of mitochondria and algae may to some extent conflict. An extensive algal symbiosis may thus be incompatible with a well-developed capacity for mitochondrial signaling. © 2008 Elsevier Ltd. All rights reserved.
Contents 1. 2. 3. 4. 5. 6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS and patterning in solitary polyps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS and patterning in colonies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS and photosynthetic symbionts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Limitations and evolutionary constraints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Mitochondria are present in one form or another in virtually all eukaryotic cells. Typically, they function in energy conversion, oxidizing substrate and using this reducing power to generate an electrochemical gradient that spans the inner mitochondrial membrane. Directly or indirectly, this gradient is used to perform the vast majority of work in the cell. An emerging consensus suggests that mitochondria, hydrogenosomes, and related organelles are vestiges of bacteria that became endosymbionts early in the history of eukaryotes [1,2]. In such early eukaryotes, selection may have favored divergent traits in host and symbiont. Likely, selection favored symbionts that could manipulate their host cells to their own advantage [3]. For instance, releasing ATP to the host would result in a faster host replication rate, thus providing more habitat for the symbionts. Alternatively, releasing reactive oxygen
∗ Tel.: +1 815 753 7899; fax: +1 815 753 0461. E-mail address: [email protected]. 1084-9521/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2008.12.006
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species (ROS) to the host might trigger sexual recombination and genetically novel host cells for the symbionts to inhabit. While such ideas remain speculative and difficult to test, they nevertheless provide a useful framework for our understanding of features found in modern eukaryotic cells such as programmed cell death [3]. The role of mitochondria in patterns and processes of development can be similarly illuminated. The ancient synergisms and antagonisms between mitochondria and their hosts may underlie the release of metabolites or metabolic by-products that in turn trigger patterns of growth, differentiation, movement, and death in host cells. At the same time, there remain important functional consequences of such metabolic signaling, thus superimposing an ever-shifting selective milieu over these putative vestiges. The premise of this review is that modern studies of the role of mitochondria in development can illuminate both historical vestiges and current selective forces. Relatively simple and in some ways primitive animals such as cnidarians may be particularly useful in such investigations. Cnidarians are basally branching animals, often termed “diploblasts” to reflect the presence of only two embryonic germ layers. Cnidarians include anthozoans (anemones and corals),
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Fig. 1. Schemata of the mitochondrial electron transport chain, showing complexes I–V, coenzyme Q, and cytochrome c. Small arrows trace the flow of electrons from NADH and FADH2 to oxygen. Large arrows show the extrusion of protons (H+ ) by complexes I, III, and IV and the return of protons to the matrix via ATP synthase (complex V), triggering the assembly of ATP (dashed arrow). Reactive oxygen species form primarily at sites A and B (modified from [39]).
hydrozoans (Hydra and other hydroids), scyphozoans (jellyfish), and other less well-known forms. With the possible exception of the enigmatic ctenophores, cnidarians are the most basally branching living animals that have a mouth and a body axis; carnivorous feeding is usually facilitated by tentacles equipped with stinging cells called cnidocytes that are used to subdue prey. Often, cnidarians alternate between a more-or-less sessile polyp stage and a swimming medusoid stage. In many cases, the polyp stage forms a colony; in such a colony, polyps are connected by a common gastrovascular system. Perhaps the best-known examples of such colonies are the reef-building corals, which are also notable because of their habitat of hosting symbiotic algae [4]. Studies of the effects of metabolism on the development of cnidarians have deep roots [5]. A favorite experiment of late-19th century biologists involved cutting the base of the stalk of the hydroid Tubularia and removing the hydranth (the mouth and tentacles) at the other end. This undifferentiated stalk could then serve as the basis for a variety of experiments. For instance, if one end was buried in sand, the other end invariably developed a hydranth, regardless of the original polarity. Various competing explanations for this and similar results were developed. Proponents of metabolic hypotheses suggested that the sand blocked the diffusion of oxygen to the buried end. A metabolic gradient thus formed and influenced the observed pattern of development. Alternatively, others suggested that a diffusible factor released from the stalk itself accumulated near the buried end and inhibited the development of a hydranth. At the time, existing methodologies did not allow distinguishing between these competing hypotheses. Such experiments did stimulate further work on metabolic gradients in cnidarians and other organisms as well. Foremost among researchers studying these gradients was Charles Manning Child. Child and his long-time collaborator, Libby Henrietta Hyman, demonstrated gradients in the effects of metabolic poisons (e.g., cyanide) and in oxygen uptake (using Winkler titrations) in a number of cnidarians, e.g. [6]. Nevertheless, by the 1940s this work fell into disrepute and was largely abandoned by developmental biologists. While some attribute the demise of Child and his theories to the rise of genetics [7], the still nascent understanding of mitochondrial bioenergetics was no doubt a contributing factor. Only in the 1960s and 1970s (e.g. [8]) was a sufficient understanding of bioenergetics developed to allow firmly grounded experimental approaches to illuminate the actual effects of mitochondria on development. In the last decade, elaboration of the role of mitochondria in programmed cell death [1] has further diminished the skepticism of main-stream developmental biologists and further opened the door to broader considerations of the effects of mitochondria. Indeed, the long-discredited views of Child and co-workers have a compelling rationale. Consider cnidarian polyps growing in the ocean. Such polyps are typically very long-lived. For instance, clones
of hydra may be immortal [9] as may be anemones and corals [10]. In the laboratory, we have used a clone of the same hydroid colony for over 15 years of experiments [11], and this is not uncommon [12]. So cnidarian polyps growing in the sea may persist for a very great while. Nevertheless, the environment certainly may vary. The polyp stage is typically sedentary, so responses to the environment must occur by growth and development or differentiation of some sort. What better signal than substrate or metabolic by-products, or both? In the case of a colony, for example, polyps that receive sufficient food can trigger greater development in these areas, while polyps that are underfed can suppress further development while nonetheless continuing to sample the same area lest it become more productive. Similar logic applies to solitary polyps which can either replicate clonally in the same area or initiate gamete production or a medusoid stage for dispersal and sexual reproduction. For instance, a solitary polyp in a rich habitat should rapidly replicate itself to take advantage of the favorable conditions. A polyp in a poor or stressful habitat, on the other hand, should initiate sex or dispersal, or both. In this way, escape from the poor or stressful habitat can be accomplished by physical dispersal or by producing novel genotypes that can better cope with the stress. Metabolic or “redox” signaling very effectively integrates environmental signals and can achieve such divergent outcomes [13,14]. In the case of mitochondria, substrate is oxidized and electrons are carried to the electron transport chain by cofactors such as NADH and FADH2 (Fig. 1). Oxidation of these cofactors leads to the sequential reduction of the electron carriers. Typically, diatomic oxygen serves as the terminal electron acceptor. However, if the electron carriers become highly reduced, some electrons may be diverted to oxygen before the final step of electron transport. This results in reactive oxygen species, broadly defined as partially reduced forms of oxygen (see [15]). If both substrate and oxygen are plentiful, and there is metabolic demand from the cell and organism, the formation of ROS is maintained at moderate levels (“state 3”). If substrate is in short supply (starvation), ROS levels are minimal (“state 2”). If metabolic demand is low, ROS levels are maximal (“state 4”). Thus a signaling system based on mitochondrial ROS can effectively monitor and distinguish among these and other divergent environmental circumstances [16]. Such considerations suggest that metabolic signaling may have a large effect on cnidarian biology. At the same time, cnidarian biology may provide useful insight into the possibilities inherent in mitochondrial signaling. In concert with studies of sponges and placozoans, considerable insight into the nature of the first animals may thus be obtained [17]. Currently, interest in coral bleaching has further stimulated studies of redox signaling in cnidarians. Such bleaching occurs when corals expel their algal symbionts. While these algae are clearly much more recent symbionts than mitochondria, and while their primary function is photosynthesis not respiration, metabolic signaling and metabolic signals (particu-
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larly ROS) nevertheless seen to be central to coral bleaching [4,18]. Thus studies of bleaching require a broader understanding of the general patterns and processes of redox signaling in cnidarians. Here the logic of mitochondrial signaling is outlined in the context of the biology of both solitary and colonial polyps, and recent studies of such signaling, for the most part focused on ROS, are reviewed. Both mitochondrial and non-mitochondrial ROS (including those from algal symbionts) are considered and integrated into the same framework. Finally, possible limitations of metabolic signaling are pointed out, and it is suggested that reliance on ROS in development may in some circumstances be subject to evolutionary constraints. In particular, host–symbiont signaling with algal ROS in anemones and corals may preclude extensive use of mitochondrial ROS in colony patterning. Conversely, utilization of mitochondrial ROS in patterning of colonial hydroids may preclude establishing algal symbiosis.
2. ROS and patterning in solitary polyps Given favorable conditions, cnidarian polyps seem capable of unlimited clonal reproduction [9]. Such favorable conditions could in part be indicated by mitochondrial redox state, e.g., state 3. Changing conditions can also be indicated by metabolic signaling, e.g., starvation (state 2) or little metabolic demand (state 4). As suggested above, in solitary polyps changing conditions typically trigger dispersal, sex, or both. Thus it would not be surprising to find that mitochondrial redox state regulates the transition between forming more polyps and forming sexual or dispersal stages. While studies of redox state have not been done in this context, there are some suggestive studies of the effects of ROS in solitary polyps. The scyphozoan Aurelia aurita is a cosmopolitan jellyfish. Likely more than one species make up this complex [19,20]. In Aurelia, the medusae form from the polyp stage by strobilation. During this process, the body of the polyp segments into several pieces with each one developing into a free-swimming medusa. As expected, environmental factors (temperature, light, and nutrition) are known to influence this process. Iodide ions seem to be necessary as well. In an important integrative synthesis, Berking et al. [21] show that in the polyps, metabolic ROS convert iodide into iodine, and it is the latter that triggers strobilation. Since mitochondria are the major source of such metabolic ROS [22], it is reasonable to expect that they provide the ROS that oxidize iodide [21]. It might be that once a polyp ceases growing, metabolic demand diminishes and mitochondria enter state 4. High rates of ROS formation then ensue, and strobilation is triggered. If mitochondria are the source of the ROS, any number of experimental perturbations could be used to examine the effects of enhanced or diminished mitochondrial ROS [23], and a corresponding effect on strobilation would be expected. While straightforward, such experiments have not yet been carried out. Other studies of ROS in solitary polyps focus on Hydra, the best-studied cnidarian model. The various species and strains of Hydra include a variety of fresh-water polyps that entirely lack a medusoid stage. In response to environmental factors (e.g., temperature), Hydra will initiate gamete production directly. Shortly after zygote formation a resting stage is typically formed which is capable of over-wintering. Although little attention has been given to either metabolic signaling or the effects of ROS on the development of these organisms, there have been some suggestive findings. In normal polyps, activity of peroxidase, an enzyme that reduces hydrogen peroxide (a form of ROS) to water, strongly localizes to certain cells located in the “foot” or base of the animal. This association is sufficiently strong so that peroxidase activity can be used as a marker for tissue of this body region [24]. In Hydra, Jantzen et al. [25] noticed that lithium treatment resulted in expression
of this characteristic peroxidase activity in various patches of tissue throughout the body column. They investigated whether this effect indicated a change in pattern formation (i.e., the upper parts of the polyp were becoming “foot-like”) or alternatively a general up-regulation of anti-oxidant enzymes in response to lithium treatment. Their data suggest that peroxide may have a role in mediating the response of Hydra to lithium, although the nature of this mediation still remains unclear. Peroxide may affect signal transduction, or may trigger cell death thus inhibiting normal pattern formation, or both. The source of this peroxide is also not clear. While Jantzen et al. [25] used cyanide (which binds to complex IV, see Fig. 1) to inhibit the electron transport chain, such inhibition will often tend to enhance the production of mitochondrial ROS (because the electron carriers become highly reduced, i.e., inhibition mimics state 4). Thus the finding of cyanide insensitivity does not necessarily rule out a mitochondrial source for ROS. The role of the Wnt signaling pathway in cnidarian patterning [26,27] can also be viewed in the context of metabolic signaling. In bilaterians, Wnt ligands activate cell-surface receptors and stabilize the cytoplasmic protein -catenin [28]. In turn, -catenin interacts with FOXO transcription factors, which are also regulated by insulin and oxidative-stress pathways [29]. Such metabolic regulation of Wnt signaling could function as a switch by which a polyp could alternate between clonal and sexual reproduction. These and similar ideas [30,31] have not been recently investigated.
3. ROS and patterning in colonies Cnidarian colonies can be long-lived, and in the case of corals, sometimes extremely long-lived. Yet the environmental circumstances under which a colony is growing can change relatively quickly even in seemingly homogenous tropical environments (e.g., following storms). Since cnidarians are typically carnivores, patterns of prey availability may be particularly important. As well, cnidarians that encrust surfaces may need to detect and integrate environmental signals that indicate when continued growth is favorable versus when resources should be allocated to gamete or medusa production (in parallel to the situation of solitary polyps). Metabolic signaling can successfully guide colonies in all of these circumstances. For instance, if the pattern of food availability shifts from one part of a colony to another, metabolic signals can be used to suppress growth and even trigger regression in the former area while stimulating growth in the latter area. Similarly, a growing colony will experience metabolic demand, shifting the redox state in the direction of oxidation (state 3). On the other hand, when the available surface is completely covered, metabolic demand will slow and the redox state will shift in the direction of reduction (state 4). Such signals could be used to trigger a shift in energy allocation from outward growth to, for instance, medusa production. The cnidarian colonies that have been the best studied for metabolic signaling are the hydractiniid hydroids. A number of features render these hydroids a useful model system [32]. Colonies can be grown on glass from either newly metamorphosed “primary” polyps or from clonal explants (small pieces of existing colonies that are surgically removed and reattached to a new surface). Adult colonies consist of feeding and reproductive polyps connected by stolons (Fig. 2). Colonies feed and contractions of the muscular polyps drive gastrovascular fluid through the stolons [33,34]. Gastrovascular flow as well as colony growth probably provides the bulk of the metabolic demand of the colony. Mitochondrion-rich cells are found at the junction of polyps and stolons [35]. These cells can be easily visualized and measured for redox state, though their uniqueness was not fully appreciated by early studies (e.g. [36]). Typically, a cell only becomes mitochondrion rich when it is subjected to high levels of metabolic demand [15]. In seagulls [37]
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Fig. 2. A hydractiniid hydroid colony encrusting an 18 mm diameter cover glass. Colony is viewed from above; polyps are bright and circular, while stolons are darker and web-like.
and fish [38], osmoregulation provides this metabolic demand. In hydractiniid hydroids it seems likely that these cells are not involved in osmoregulation, but rather regulate gastrovascular flow. All of the following suggest such a role for cnidarian mitochondrionrich cells: their presence at the junction between the muscular polyp and the non-muscular stolon [35]; their apparent absence in cnidarians that largely use cilia and not muscular contractions to propel flow (unpublished data); the correspondence of their redox states to maximal metabolic demand at times of maximal flow and conversely [39]. In species of hydractiniid hydroids, workto-date suggests that the mitochondrion-rich region functions as a valve between the polyp and the rest of the colony (Fig. 3). When mitochondrion-rich cells relax, the valve closes; when these cells contract, the valve opens. While during the feeding response the valve is closed, shortly after feeding the valve opens in concert with polyp contractions, and food is distributed throughout the colony by the gastrovascular fluid. This contractile function of mitochondrionrich cells may be an important factor in synchronizing redox-related emissions. In particular, mitochondrion-rich cells and their metabolic states may provide crucial regulation of ROS emissions by the colony. Such emissions can be easily visualized by fluorescent markers (Fig. 4). Redox state of these cells can be visualized by native fluorescence of NAD(P)H. A number of studies [11,23,35,36,39–41] suggest the following view of how metabolic signaling can be integrated into adaptive colony development. Consider a colony encrusting
Fig. 3. Schematic of longitudinal section showing the hypothesized function of mitochondrion-rich cells in cnidarians. Successful feeding is followed by contractions of these cells (A). In concert with the musculature and hydrostatic skeleton of the polyp (above) and the rigid perisarc of the stolon (below), these contractions draw the band of tissue at the base tighter, opening the lumen, and allowing foodrich gastrovascular fluid to enter the stolon. When these mitochondrion-rich cells cease contracting (B), the tissue relaxes, closing the lumen.
Fig. 4. Photomicrograph of a transverse section of the base of a polyp of a hydractiniid hydroid stained with H2 DCFDA. Colony is growing on cover glass and is viewed from below with an inverted microscope. Once acetate groups are removed by intracellular esterases, H2 DCF can be oxidized by reactive oxygen species. The resulting fluorescence indicates that this oxidation occurs in mitochondrion-rich epitheliomuscular cells (the bright circular areas; each area is ≈5 m in diameter).
a hermit crab shell, a typical habitat for hydractiniid hydroids: given the vagaries of hermit crab behavior and resulting water currents, some polyps may feed while others do not. In areas of a colony that do not directly feed, polyps undergo minimal contractions, and there is little metabolic demand. Nevertheless, these areas of the colony are supplied with substrate via stolons from well-fed polyps. In unfed polyps there is thus sufficient substrate and little metabolic demand, and mitochondria (particularly those of mitochondrion-rich cells) enter the resting state (state 4). ROS emissions are maximal, and these ROS may suppress further local development of polyps and stolons. On the other hand, polyps in areas of the colony that are well fed contract maximally. Metabolic demand is maximal, and the redox state of mitochondria, particularly those in mitochondrion-rich cells, shifts in the direction of oxidation. ROS emissions are moderate and further development of polyps and stolons is no longer inhibited. Hence the colony develops in areas where food resources are most abundant. Interestingly, it is the behavior of the mitochondrion-rich cells (whether they are contracting or not) that determines their redox state. At the level of a cell, addition of substrate would typically shift the redox state in the direction of reduction. However, because feeding triggers gastrovascular flow [34], in this system addition of substrate triggers contractions and metabolic demand in the mitochondrion-rich cells. The result is a shift of their redox state in the direction of oxidation, the opposite of simple, cell-level predictions. Regulation of redox state thus occurs at the level of the group of cells, and such regulation is influenced by the cells’ grouplevel task. Since the observed response is distinct from that of single cells, this has been termed “multicellular redox regulation” [41,42]. While the above-integrated view is supported by a number of experiments, a “many pathways” view of ROS is emerging depending on where in the colony ROS are found and at what levels [40]. Typically, the mitochondrion-rich cells show the highest levels of ROS (Fig. 4). Polyps also show moderate levels, while stolons are usually completely devoid of ROS with the exception of low levels near the tip (Fig. 5). Nevertheless, if a colony experiences
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Fig. 5. Photomicrograph of a stolon tip of a hydractiniid hydroid (≈30 m in diameter). Colony is growing on cover glass and is viewed from below with an inverted microscope. After the colony was treated with a nitric oxide donor and stained with H2 DCFDA, the stolon tip produced high levels of reactive oxygen species and regressed.
stress, colony-wide gastrovascular flow tends to diminish or cease. Depending on the severity of the stress and the extent to which gastrovascular flow is diminished, ROS and related reactive nitrogen species (RNS, e.g., nitric oxide) will then appear in some or all of the stolon tips (Fig. 5). It is not known if the source of these ROS and RNS is mitochondrial. Subsequently, the affected stolon tips will regress [11]. This is a particular example of what seems to be generally found in cnidarians and other simple organisms as well. As described by Buss [43]: “Clonal organisms typically possess one or more clonewide fluid-conducting systems. . .. The functioning of these systems will generate local patterns. . .. If such features can be detected and the signals transduced to effect expression of patternforming genes, global rules responsive to local state arise.” ROS thus seem capable of inhibiting growth at moderate levels while actually triggering regression at still higher levels. Both cases, however, tend to result in adaptive growth and development: on the one hand, poor micro-habitats suppress proliferation of polyps, while on the other especially unfavorable habitats trigger active regression and centralized growth in the more favorable core area. An association between medusa production and metabolic byproducts is suggested by the work of Braverman [44,45]. Braverman treated colonies of a hydractiniid hydroid with carbon dioxide (the end product of aerobic oxidation of substrate) and found that a greater number of polyps developed medusae than in untreated controls. While Braverman argues for a direct effect, it is also possible that an increased concentration of CO2 has a number of indirect effects (e.g., on pH and dissolved calcium). Thus CO2 may actually trigger a more generalized stress response. However, earlier workers [30] and indeed Child himself devised a number of ingenious experiments that may argue against a general response. Other data suggest that gamete production may be enhanced by metabolites of the mitochondrial citrate cycle and that nutritive and reproductive polyps may have different enzymatic profiles from feeding polyps [46]. Thus a connection between metabolism and sexual development may exist in these hydroids, although further work is needed to clarify the nature of this connection. 4. ROS and photosynthetic symbionts Much of the current interest in metabolic signaling in cnidarians focuses not on mitochondria, but on the much more recently acquired endosymbionts, Symbiodidium algae, which are also called zooxanthellae. These algae are hosted by a number of corals and anemones. Such more recent symbioses may employ some of the pre-existing mitochondrial pathways, or may otherwise influence or conflict with mitochondrial signaling, so these algae are worth considering in this context. As well, the considerable interest in coral reefs is highly justified. Coral reefs are among the most biologically diverse ecosystems on earth, containing up to a quarter
Fig. 6. Two genetically identical colonies of a gorgonian octocoral are shown. The smaller colony in the foreground has bleached, while the large colony in the background has retained its symbionts. Polyps in the foreground are ≈5 mm tall.
of all marine species. At the same time, these reefs provide vital ecosystem services to human society, including fisheries, coastal protection, building materials, new biochemical compounds, and tourism. Nevertheless, coral reefs are currently threatened by the poorly understood process of coral bleaching, in which corals release their symbiotic algae and appear pale and “bleached” (Fig. 6) [47,48]. The algae symbiosis dominates the biology of their cnidarian hosts. Indeed, such hosts can be viewed as a chimera between alga and animal. Many features of corals and anemones – absorption and concentration of inorganic carbon and various forms of nitrogen, tolerance to damaging sunlight, well-developed anti-oxidant defenses, and so on – facilitate and protect the symbionts [4]. The algae in turn provide the animal host with the products of photosynthesis. It is likely that the host and symbiont form an extensive web of metabolic interactions; bleaching may result from the partial or complete disruption of this web of metabolic signaling. Algae produce ROS which can contribute to photo-oxidative stress in the host [49,50]. Anti-oxidant capabilities of the host likely developed in this context [4,51–53]. At the same time, the host itself seems to produce high levels of RNS as a sort of “eviction notice” during the bleaching process [18]. Multiple mechanisms may thus be involved in bleaching culminating in host-cell detachment, expulsion through the host-cell membrane (exocytosis), digestion, and cell death [54]. For the most part, laboratory studies have focused on anemones because they are easier to culture. Corals have been primarily studied in the field, although notable laboratory studies have also been done [55,56]. Anemones, however, lack crucial features of corals: they are not colonial and thus do not have connections between polyps—a colonial gastrovascular system. Such a gastrovascular system may mediate bleaching by moving detached host cells and zooxanthellae released by exocytosis through and ultimately out of the colony [57]. The effects of temperature on a colonial gastrovascular system have been studied in hydractiniid hydroids [58]. Higher temperatures induce chaotic gastrovascular dynamics, and such dynamics may underlie bleaching episodes. Hydractiniid hydroids, however, lack algal symbionts and therefore do not bleach. Crucial studies of the gastrovascular system of corals have thus been hindered by the absence of a sufficiently tractable laboratory model. 5. Limitations and evolutionary constraints While metabolic signaling has many advantages particularly in long-lived organisms, it also has some disadvantages [17].
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Metabolic signals may under some circumstances lack specificity. Consider the lack of oxygen and the lack of metabolic demand: both will shift the redox state of mitochondria in the direction of reduction. The metabolic signals will thus be similar, even as the necessary responses are very different. While incorporating temporal dynamics into the signaling mechanism may provide a solution to this particular problem, the general point nevertheless remains. Consider for instance a cnidarian host subject to metabolic signaling from both mitochondria and algae. In some instances, the subcellular localization of ROS may help distinguish between the two sources. However, if the signaling occurs between mitochondrionrich cells and neighboring cells as described in Section 3, subcellular localization of the organelles would likely not provide effective discrimination. In such a case, there may be little possibility for a signaling mechanism that distinguishes ROS from mitochondria and ROS from algae. The use of the ROS in signaling from one of these sources may thus largely preclude the use of ROS from the other source. This may have served as a constraint in the evolution of both anthozoans (anemones and corals) and hydrozoans (including Hydra and the hydractiniid hydroids). Consider that representatives of the anthozoan sister group (the medusozoans, which include hydrozoans and scyphozoans) employ muscular activity both in the swimming of medusae [59] and in the circulation of gastrovascular fluid as described above in Section 3. While anthozoans certainly use muscles to retract, this may be a relatively simple activity compared to using co-ordinated colony-wide muscular contractions to propel gastrovascular fluid. Indeed, anthozoans typically exhibit cilia-driven gastrovascular flow [60]. Without a sophisticated nervous system, accomplishing muscle-driven flow may require mitochondrial signaling. Corals and anemones may not be able to do this, because their algal symbionts rely on the same metabolic pathways as mitochondria. Thus such host–symbiont signaling may have served as an important constraint in anthozoan evolution. At the same time, hydroids that use muscular contractions to drive gastrovascular flow may be precluded from establishing lasting symbioses with algae. Indeed, the best-known hydroids with photosynthetic symbionts are the “green hydras.” Since these are not colonial, they do not have sophisticated patterns of muscle-driven gastrovascular flow. While there are colonial hydroids with Symbiodinium symbionts [61], the characteristics of the gastrovascular flow in these colonies remain unknown. Exploring metabolic signaling in such symbiotic colonial hydroids would provide an obvious test of this hypothesis. 6. Conclusions Study of metabolic signaling in cnidarians has a rich history, and there is ample evidence that such an approach can significantly illuminate the biology of these basally branching organisms. In particular, mitochondrial ROS appear to have significant signaling functions in both solitary and colonial polyp stages. In parallel, coral bleaching involves the disruption of metabolic signaling between the host and algal symbionts. Viewing both mitochondrial and algal signaling within the same integrated framework will enhance our understanding of both of these processes. Acknowledgement This work is supported by the NSF (IBN-0090580 and EF0531654). References [1] Lane N. Power, sex, suicide: mitochondria and the meaning of life. Oxford: Oxford University Press; 2005.
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Contents lists available at ScienceDirect
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Review
Mitochondria and the redox control of development in cnidarians Neil Blackstone ∗ Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115, USA
a r t i c l e
i n f o
Article history: Available online 24 December 2008 Keywords: Coral bleaching Cnidarian Mitochondrion-rich cell Reactive oxygen species Redox signaling
a b s t r a c t Mitochondria are the product of an ancient symbiosis between bacteria and host cells. While mitochondria function primarily in energy conversion, increasing amounts of evidence indicate that mitochondrial metabolic state can influence various emergent features of eukaryotic cells. Important intermediaries in such redox signaling include by-products of metabolism, particularly reactive oxygen species (ROS). This review uses cnidarians, a group of basally branching animals, to illustrate the many and varied effects of ROS on development. ROS from both mitochondria and algal symbionts are considered. Because some applications of ROS may lack specificity, the signaling demands of mitochondria and algae may to some extent conflict. An extensive algal symbiosis may thus be incompatible with a well-developed capacity for mitochondrial signaling. © 2008 Elsevier Ltd. All rights reserved.
Contents 1. 2. 3. 4. 5. 6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS and patterning in solitary polyps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS and patterning in colonies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS and photosynthetic symbionts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Limitations and evolutionary constraints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Mitochondria are present in one form or another in virtually all eukaryotic cells. Typically, they function in energy conversion, oxidizing substrate and using this reducing power to generate an electrochemical gradient that spans the inner mitochondrial membrane. Directly or indirectly, this gradient is used to perform the vast majority of work in the cell. An emerging consensus suggests that mitochondria, hydrogenosomes, and related organelles are vestiges of bacteria that became endosymbionts early in the history of eukaryotes [1,2]. In such early eukaryotes, selection may have favored divergent traits in host and symbiont. Likely, selection favored symbionts that could manipulate their host cells to their own advantage [3]. For instance, releasing ATP to the host would result in a faster host replication rate, thus providing more habitat for the symbionts. Alternatively, releasing reactive oxygen
∗ Tel.: +1 815 753 7899; fax: +1 815 753 0461. E-mail address: [email protected]. 1084-9521/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2008.12.006
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species (ROS) to the host might trigger sexual recombination and genetically novel host cells for the symbionts to inhabit. While such ideas remain speculative and difficult to test, they nevertheless provide a useful framework for our understanding of features found in modern eukaryotic cells such as programmed cell death [3]. The role of mitochondria in patterns and processes of development can be similarly illuminated. The ancient synergisms and antagonisms between mitochondria and their hosts may underlie the release of metabolites or metabolic by-products that in turn trigger patterns of growth, differentiation, movement, and death in host cells. At the same time, there remain important functional consequences of such metabolic signaling, thus superimposing an ever-shifting selective milieu over these putative vestiges. The premise of this review is that modern studies of the role of mitochondria in development can illuminate both historical vestiges and current selective forces. Relatively simple and in some ways primitive animals such as cnidarians may be particularly useful in such investigations. Cnidarians are basally branching animals, often termed “diploblasts” to reflect the presence of only two embryonic germ layers. Cnidarians include anthozoans (anemones and corals),
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Fig. 1. Schemata of the mitochondrial electron transport chain, showing complexes I–V, coenzyme Q, and cytochrome c. Small arrows trace the flow of electrons from NADH and FADH2 to oxygen. Large arrows show the extrusion of protons (H+ ) by complexes I, III, and IV and the return of protons to the matrix via ATP synthase (complex V), triggering the assembly of ATP (dashed arrow). Reactive oxygen species form primarily at sites A and B (modified from [39]).
hydrozoans (Hydra and other hydroids), scyphozoans (jellyfish), and other less well-known forms. With the possible exception of the enigmatic ctenophores, cnidarians are the most basally branching living animals that have a mouth and a body axis; carnivorous feeding is usually facilitated by tentacles equipped with stinging cells called cnidocytes that are used to subdue prey. Often, cnidarians alternate between a more-or-less sessile polyp stage and a swimming medusoid stage. In many cases, the polyp stage forms a colony; in such a colony, polyps are connected by a common gastrovascular system. Perhaps the best-known examples of such colonies are the reef-building corals, which are also notable because of their habitat of hosting symbiotic algae [4]. Studies of the effects of metabolism on the development of cnidarians have deep roots [5]. A favorite experiment of late-19th century biologists involved cutting the base of the stalk of the hydroid Tubularia and removing the hydranth (the mouth and tentacles) at the other end. This undifferentiated stalk could then serve as the basis for a variety of experiments. For instance, if one end was buried in sand, the other end invariably developed a hydranth, regardless of the original polarity. Various competing explanations for this and similar results were developed. Proponents of metabolic hypotheses suggested that the sand blocked the diffusion of oxygen to the buried end. A metabolic gradient thus formed and influenced the observed pattern of development. Alternatively, others suggested that a diffusible factor released from the stalk itself accumulated near the buried end and inhibited the development of a hydranth. At the time, existing methodologies did not allow distinguishing between these competing hypotheses. Such experiments did stimulate further work on metabolic gradients in cnidarians and other organisms as well. Foremost among researchers studying these gradients was Charles Manning Child. Child and his long-time collaborator, Libby Henrietta Hyman, demonstrated gradients in the effects of metabolic poisons (e.g., cyanide) and in oxygen uptake (using Winkler titrations) in a number of cnidarians, e.g. [6]. Nevertheless, by the 1940s this work fell into disrepute and was largely abandoned by developmental biologists. While some attribute the demise of Child and his theories to the rise of genetics [7], the still nascent understanding of mitochondrial bioenergetics was no doubt a contributing factor. Only in the 1960s and 1970s (e.g. [8]) was a sufficient understanding of bioenergetics developed to allow firmly grounded experimental approaches to illuminate the actual effects of mitochondria on development. In the last decade, elaboration of the role of mitochondria in programmed cell death [1] has further diminished the skepticism of main-stream developmental biologists and further opened the door to broader considerations of the effects of mitochondria. Indeed, the long-discredited views of Child and co-workers have a compelling rationale. Consider cnidarian polyps growing in the ocean. Such polyps are typically very long-lived. For instance, clones
of hydra may be immortal [9] as may be anemones and corals [10]. In the laboratory, we have used a clone of the same hydroid colony for over 15 years of experiments [11], and this is not uncommon [12]. So cnidarian polyps growing in the sea may persist for a very great while. Nevertheless, the environment certainly may vary. The polyp stage is typically sedentary, so responses to the environment must occur by growth and development or differentiation of some sort. What better signal than substrate or metabolic by-products, or both? In the case of a colony, for example, polyps that receive sufficient food can trigger greater development in these areas, while polyps that are underfed can suppress further development while nonetheless continuing to sample the same area lest it become more productive. Similar logic applies to solitary polyps which can either replicate clonally in the same area or initiate gamete production or a medusoid stage for dispersal and sexual reproduction. For instance, a solitary polyp in a rich habitat should rapidly replicate itself to take advantage of the favorable conditions. A polyp in a poor or stressful habitat, on the other hand, should initiate sex or dispersal, or both. In this way, escape from the poor or stressful habitat can be accomplished by physical dispersal or by producing novel genotypes that can better cope with the stress. Metabolic or “redox” signaling very effectively integrates environmental signals and can achieve such divergent outcomes [13,14]. In the case of mitochondria, substrate is oxidized and electrons are carried to the electron transport chain by cofactors such as NADH and FADH2 (Fig. 1). Oxidation of these cofactors leads to the sequential reduction of the electron carriers. Typically, diatomic oxygen serves as the terminal electron acceptor. However, if the electron carriers become highly reduced, some electrons may be diverted to oxygen before the final step of electron transport. This results in reactive oxygen species, broadly defined as partially reduced forms of oxygen (see [15]). If both substrate and oxygen are plentiful, and there is metabolic demand from the cell and organism, the formation of ROS is maintained at moderate levels (“state 3”). If substrate is in short supply (starvation), ROS levels are minimal (“state 2”). If metabolic demand is low, ROS levels are maximal (“state 4”). Thus a signaling system based on mitochondrial ROS can effectively monitor and distinguish among these and other divergent environmental circumstances [16]. Such considerations suggest that metabolic signaling may have a large effect on cnidarian biology. At the same time, cnidarian biology may provide useful insight into the possibilities inherent in mitochondrial signaling. In concert with studies of sponges and placozoans, considerable insight into the nature of the first animals may thus be obtained [17]. Currently, interest in coral bleaching has further stimulated studies of redox signaling in cnidarians. Such bleaching occurs when corals expel their algal symbionts. While these algae are clearly much more recent symbionts than mitochondria, and while their primary function is photosynthesis not respiration, metabolic signaling and metabolic signals (particu-
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larly ROS) nevertheless seen to be central to coral bleaching [4,18]. Thus studies of bleaching require a broader understanding of the general patterns and processes of redox signaling in cnidarians. Here the logic of mitochondrial signaling is outlined in the context of the biology of both solitary and colonial polyps, and recent studies of such signaling, for the most part focused on ROS, are reviewed. Both mitochondrial and non-mitochondrial ROS (including those from algal symbionts) are considered and integrated into the same framework. Finally, possible limitations of metabolic signaling are pointed out, and it is suggested that reliance on ROS in development may in some circumstances be subject to evolutionary constraints. In particular, host–symbiont signaling with algal ROS in anemones and corals may preclude extensive use of mitochondrial ROS in colony patterning. Conversely, utilization of mitochondrial ROS in patterning of colonial hydroids may preclude establishing algal symbiosis.
2. ROS and patterning in solitary polyps Given favorable conditions, cnidarian polyps seem capable of unlimited clonal reproduction [9]. Such favorable conditions could in part be indicated by mitochondrial redox state, e.g., state 3. Changing conditions can also be indicated by metabolic signaling, e.g., starvation (state 2) or little metabolic demand (state 4). As suggested above, in solitary polyps changing conditions typically trigger dispersal, sex, or both. Thus it would not be surprising to find that mitochondrial redox state regulates the transition between forming more polyps and forming sexual or dispersal stages. While studies of redox state have not been done in this context, there are some suggestive studies of the effects of ROS in solitary polyps. The scyphozoan Aurelia aurita is a cosmopolitan jellyfish. Likely more than one species make up this complex [19,20]. In Aurelia, the medusae form from the polyp stage by strobilation. During this process, the body of the polyp segments into several pieces with each one developing into a free-swimming medusa. As expected, environmental factors (temperature, light, and nutrition) are known to influence this process. Iodide ions seem to be necessary as well. In an important integrative synthesis, Berking et al. [21] show that in the polyps, metabolic ROS convert iodide into iodine, and it is the latter that triggers strobilation. Since mitochondria are the major source of such metabolic ROS [22], it is reasonable to expect that they provide the ROS that oxidize iodide [21]. It might be that once a polyp ceases growing, metabolic demand diminishes and mitochondria enter state 4. High rates of ROS formation then ensue, and strobilation is triggered. If mitochondria are the source of the ROS, any number of experimental perturbations could be used to examine the effects of enhanced or diminished mitochondrial ROS [23], and a corresponding effect on strobilation would be expected. While straightforward, such experiments have not yet been carried out. Other studies of ROS in solitary polyps focus on Hydra, the best-studied cnidarian model. The various species and strains of Hydra include a variety of fresh-water polyps that entirely lack a medusoid stage. In response to environmental factors (e.g., temperature), Hydra will initiate gamete production directly. Shortly after zygote formation a resting stage is typically formed which is capable of over-wintering. Although little attention has been given to either metabolic signaling or the effects of ROS on the development of these organisms, there have been some suggestive findings. In normal polyps, activity of peroxidase, an enzyme that reduces hydrogen peroxide (a form of ROS) to water, strongly localizes to certain cells located in the “foot” or base of the animal. This association is sufficiently strong so that peroxidase activity can be used as a marker for tissue of this body region [24]. In Hydra, Jantzen et al. [25] noticed that lithium treatment resulted in expression
of this characteristic peroxidase activity in various patches of tissue throughout the body column. They investigated whether this effect indicated a change in pattern formation (i.e., the upper parts of the polyp were becoming “foot-like”) or alternatively a general up-regulation of anti-oxidant enzymes in response to lithium treatment. Their data suggest that peroxide may have a role in mediating the response of Hydra to lithium, although the nature of this mediation still remains unclear. Peroxide may affect signal transduction, or may trigger cell death thus inhibiting normal pattern formation, or both. The source of this peroxide is also not clear. While Jantzen et al. [25] used cyanide (which binds to complex IV, see Fig. 1) to inhibit the electron transport chain, such inhibition will often tend to enhance the production of mitochondrial ROS (because the electron carriers become highly reduced, i.e., inhibition mimics state 4). Thus the finding of cyanide insensitivity does not necessarily rule out a mitochondrial source for ROS. The role of the Wnt signaling pathway in cnidarian patterning [26,27] can also be viewed in the context of metabolic signaling. In bilaterians, Wnt ligands activate cell-surface receptors and stabilize the cytoplasmic protein -catenin [28]. In turn, -catenin interacts with FOXO transcription factors, which are also regulated by insulin and oxidative-stress pathways [29]. Such metabolic regulation of Wnt signaling could function as a switch by which a polyp could alternate between clonal and sexual reproduction. These and similar ideas [30,31] have not been recently investigated.
3. ROS and patterning in colonies Cnidarian colonies can be long-lived, and in the case of corals, sometimes extremely long-lived. Yet the environmental circumstances under which a colony is growing can change relatively quickly even in seemingly homogenous tropical environments (e.g., following storms). Since cnidarians are typically carnivores, patterns of prey availability may be particularly important. As well, cnidarians that encrust surfaces may need to detect and integrate environmental signals that indicate when continued growth is favorable versus when resources should be allocated to gamete or medusa production (in parallel to the situation of solitary polyps). Metabolic signaling can successfully guide colonies in all of these circumstances. For instance, if the pattern of food availability shifts from one part of a colony to another, metabolic signals can be used to suppress growth and even trigger regression in the former area while stimulating growth in the latter area. Similarly, a growing colony will experience metabolic demand, shifting the redox state in the direction of oxidation (state 3). On the other hand, when the available surface is completely covered, metabolic demand will slow and the redox state will shift in the direction of reduction (state 4). Such signals could be used to trigger a shift in energy allocation from outward growth to, for instance, medusa production. The cnidarian colonies that have been the best studied for metabolic signaling are the hydractiniid hydroids. A number of features render these hydroids a useful model system [32]. Colonies can be grown on glass from either newly metamorphosed “primary” polyps or from clonal explants (small pieces of existing colonies that are surgically removed and reattached to a new surface). Adult colonies consist of feeding and reproductive polyps connected by stolons (Fig. 2). Colonies feed and contractions of the muscular polyps drive gastrovascular fluid through the stolons [33,34]. Gastrovascular flow as well as colony growth probably provides the bulk of the metabolic demand of the colony. Mitochondrion-rich cells are found at the junction of polyps and stolons [35]. These cells can be easily visualized and measured for redox state, though their uniqueness was not fully appreciated by early studies (e.g. [36]). Typically, a cell only becomes mitochondrion rich when it is subjected to high levels of metabolic demand [15]. In seagulls [37]
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Fig. 2. A hydractiniid hydroid colony encrusting an 18 mm diameter cover glass. Colony is viewed from above; polyps are bright and circular, while stolons are darker and web-like.
and fish [38], osmoregulation provides this metabolic demand. In hydractiniid hydroids it seems likely that these cells are not involved in osmoregulation, but rather regulate gastrovascular flow. All of the following suggest such a role for cnidarian mitochondrionrich cells: their presence at the junction between the muscular polyp and the non-muscular stolon [35]; their apparent absence in cnidarians that largely use cilia and not muscular contractions to propel flow (unpublished data); the correspondence of their redox states to maximal metabolic demand at times of maximal flow and conversely [39]. In species of hydractiniid hydroids, workto-date suggests that the mitochondrion-rich region functions as a valve between the polyp and the rest of the colony (Fig. 3). When mitochondrion-rich cells relax, the valve closes; when these cells contract, the valve opens. While during the feeding response the valve is closed, shortly after feeding the valve opens in concert with polyp contractions, and food is distributed throughout the colony by the gastrovascular fluid. This contractile function of mitochondrionrich cells may be an important factor in synchronizing redox-related emissions. In particular, mitochondrion-rich cells and their metabolic states may provide crucial regulation of ROS emissions by the colony. Such emissions can be easily visualized by fluorescent markers (Fig. 4). Redox state of these cells can be visualized by native fluorescence of NAD(P)H. A number of studies [11,23,35,36,39–41] suggest the following view of how metabolic signaling can be integrated into adaptive colony development. Consider a colony encrusting
Fig. 3. Schematic of longitudinal section showing the hypothesized function of mitochondrion-rich cells in cnidarians. Successful feeding is followed by contractions of these cells (A). In concert with the musculature and hydrostatic skeleton of the polyp (above) and the rigid perisarc of the stolon (below), these contractions draw the band of tissue at the base tighter, opening the lumen, and allowing foodrich gastrovascular fluid to enter the stolon. When these mitochondrion-rich cells cease contracting (B), the tissue relaxes, closing the lumen.
Fig. 4. Photomicrograph of a transverse section of the base of a polyp of a hydractiniid hydroid stained with H2 DCFDA. Colony is growing on cover glass and is viewed from below with an inverted microscope. Once acetate groups are removed by intracellular esterases, H2 DCF can be oxidized by reactive oxygen species. The resulting fluorescence indicates that this oxidation occurs in mitochondrion-rich epitheliomuscular cells (the bright circular areas; each area is ≈5 m in diameter).
a hermit crab shell, a typical habitat for hydractiniid hydroids: given the vagaries of hermit crab behavior and resulting water currents, some polyps may feed while others do not. In areas of a colony that do not directly feed, polyps undergo minimal contractions, and there is little metabolic demand. Nevertheless, these areas of the colony are supplied with substrate via stolons from well-fed polyps. In unfed polyps there is thus sufficient substrate and little metabolic demand, and mitochondria (particularly those of mitochondrion-rich cells) enter the resting state (state 4). ROS emissions are maximal, and these ROS may suppress further local development of polyps and stolons. On the other hand, polyps in areas of the colony that are well fed contract maximally. Metabolic demand is maximal, and the redox state of mitochondria, particularly those in mitochondrion-rich cells, shifts in the direction of oxidation. ROS emissions are moderate and further development of polyps and stolons is no longer inhibited. Hence the colony develops in areas where food resources are most abundant. Interestingly, it is the behavior of the mitochondrion-rich cells (whether they are contracting or not) that determines their redox state. At the level of a cell, addition of substrate would typically shift the redox state in the direction of reduction. However, because feeding triggers gastrovascular flow [34], in this system addition of substrate triggers contractions and metabolic demand in the mitochondrion-rich cells. The result is a shift of their redox state in the direction of oxidation, the opposite of simple, cell-level predictions. Regulation of redox state thus occurs at the level of the group of cells, and such regulation is influenced by the cells’ grouplevel task. Since the observed response is distinct from that of single cells, this has been termed “multicellular redox regulation” [41,42]. While the above-integrated view is supported by a number of experiments, a “many pathways” view of ROS is emerging depending on where in the colony ROS are found and at what levels [40]. Typically, the mitochondrion-rich cells show the highest levels of ROS (Fig. 4). Polyps also show moderate levels, while stolons are usually completely devoid of ROS with the exception of low levels near the tip (Fig. 5). Nevertheless, if a colony experiences
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Fig. 5. Photomicrograph of a stolon tip of a hydractiniid hydroid (≈30 m in diameter). Colony is growing on cover glass and is viewed from below with an inverted microscope. After the colony was treated with a nitric oxide donor and stained with H2 DCFDA, the stolon tip produced high levels of reactive oxygen species and regressed.
stress, colony-wide gastrovascular flow tends to diminish or cease. Depending on the severity of the stress and the extent to which gastrovascular flow is diminished, ROS and related reactive nitrogen species (RNS, e.g., nitric oxide) will then appear in some or all of the stolon tips (Fig. 5). It is not known if the source of these ROS and RNS is mitochondrial. Subsequently, the affected stolon tips will regress [11]. This is a particular example of what seems to be generally found in cnidarians and other simple organisms as well. As described by Buss [43]: “Clonal organisms typically possess one or more clonewide fluid-conducting systems. . .. The functioning of these systems will generate local patterns. . .. If such features can be detected and the signals transduced to effect expression of patternforming genes, global rules responsive to local state arise.” ROS thus seem capable of inhibiting growth at moderate levels while actually triggering regression at still higher levels. Both cases, however, tend to result in adaptive growth and development: on the one hand, poor micro-habitats suppress proliferation of polyps, while on the other especially unfavorable habitats trigger active regression and centralized growth in the more favorable core area. An association between medusa production and metabolic byproducts is suggested by the work of Braverman [44,45]. Braverman treated colonies of a hydractiniid hydroid with carbon dioxide (the end product of aerobic oxidation of substrate) and found that a greater number of polyps developed medusae than in untreated controls. While Braverman argues for a direct effect, it is also possible that an increased concentration of CO2 has a number of indirect effects (e.g., on pH and dissolved calcium). Thus CO2 may actually trigger a more generalized stress response. However, earlier workers [30] and indeed Child himself devised a number of ingenious experiments that may argue against a general response. Other data suggest that gamete production may be enhanced by metabolites of the mitochondrial citrate cycle and that nutritive and reproductive polyps may have different enzymatic profiles from feeding polyps [46]. Thus a connection between metabolism and sexual development may exist in these hydroids, although further work is needed to clarify the nature of this connection. 4. ROS and photosynthetic symbionts Much of the current interest in metabolic signaling in cnidarians focuses not on mitochondria, but on the much more recently acquired endosymbionts, Symbiodidium algae, which are also called zooxanthellae. These algae are hosted by a number of corals and anemones. Such more recent symbioses may employ some of the pre-existing mitochondrial pathways, or may otherwise influence or conflict with mitochondrial signaling, so these algae are worth considering in this context. As well, the considerable interest in coral reefs is highly justified. Coral reefs are among the most biologically diverse ecosystems on earth, containing up to a quarter
Fig. 6. Two genetically identical colonies of a gorgonian octocoral are shown. The smaller colony in the foreground has bleached, while the large colony in the background has retained its symbionts. Polyps in the foreground are ≈5 mm tall.
of all marine species. At the same time, these reefs provide vital ecosystem services to human society, including fisheries, coastal protection, building materials, new biochemical compounds, and tourism. Nevertheless, coral reefs are currently threatened by the poorly understood process of coral bleaching, in which corals release their symbiotic algae and appear pale and “bleached” (Fig. 6) [47,48]. The algae symbiosis dominates the biology of their cnidarian hosts. Indeed, such hosts can be viewed as a chimera between alga and animal. Many features of corals and anemones – absorption and concentration of inorganic carbon and various forms of nitrogen, tolerance to damaging sunlight, well-developed anti-oxidant defenses, and so on – facilitate and protect the symbionts [4]. The algae in turn provide the animal host with the products of photosynthesis. It is likely that the host and symbiont form an extensive web of metabolic interactions; bleaching may result from the partial or complete disruption of this web of metabolic signaling. Algae produce ROS which can contribute to photo-oxidative stress in the host [49,50]. Anti-oxidant capabilities of the host likely developed in this context [4,51–53]. At the same time, the host itself seems to produce high levels of RNS as a sort of “eviction notice” during the bleaching process [18]. Multiple mechanisms may thus be involved in bleaching culminating in host-cell detachment, expulsion through the host-cell membrane (exocytosis), digestion, and cell death [54]. For the most part, laboratory studies have focused on anemones because they are easier to culture. Corals have been primarily studied in the field, although notable laboratory studies have also been done [55,56]. Anemones, however, lack crucial features of corals: they are not colonial and thus do not have connections between polyps—a colonial gastrovascular system. Such a gastrovascular system may mediate bleaching by moving detached host cells and zooxanthellae released by exocytosis through and ultimately out of the colony [57]. The effects of temperature on a colonial gastrovascular system have been studied in hydractiniid hydroids [58]. Higher temperatures induce chaotic gastrovascular dynamics, and such dynamics may underlie bleaching episodes. Hydractiniid hydroids, however, lack algal symbionts and therefore do not bleach. Crucial studies of the gastrovascular system of corals have thus been hindered by the absence of a sufficiently tractable laboratory model. 5. Limitations and evolutionary constraints While metabolic signaling has many advantages particularly in long-lived organisms, it also has some disadvantages [17].
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Metabolic signals may under some circumstances lack specificity. Consider the lack of oxygen and the lack of metabolic demand: both will shift the redox state of mitochondria in the direction of reduction. The metabolic signals will thus be similar, even as the necessary responses are very different. While incorporating temporal dynamics into the signaling mechanism may provide a solution to this particular problem, the general point nevertheless remains. Consider for instance a cnidarian host subject to metabolic signaling from both mitochondria and algae. In some instances, the subcellular localization of ROS may help distinguish between the two sources. However, if the signaling occurs between mitochondrionrich cells and neighboring cells as described in Section 3, subcellular localization of the organelles would likely not provide effective discrimination. In such a case, there may be little possibility for a signaling mechanism that distinguishes ROS from mitochondria and ROS from algae. The use of the ROS in signaling from one of these sources may thus largely preclude the use of ROS from the other source. This may have served as a constraint in the evolution of both anthozoans (anemones and corals) and hydrozoans (including Hydra and the hydractiniid hydroids). Consider that representatives of the anthozoan sister group (the medusozoans, which include hydrozoans and scyphozoans) employ muscular activity both in the swimming of medusae [59] and in the circulation of gastrovascular fluid as described above in Section 3. While anthozoans certainly use muscles to retract, this may be a relatively simple activity compared to using co-ordinated colony-wide muscular contractions to propel gastrovascular fluid. Indeed, anthozoans typically exhibit cilia-driven gastrovascular flow [60]. Without a sophisticated nervous system, accomplishing muscle-driven flow may require mitochondrial signaling. Corals and anemones may not be able to do this, because their algal symbionts rely on the same metabolic pathways as mitochondria. Thus such host–symbiont signaling may have served as an important constraint in anthozoan evolution. At the same time, hydroids that use muscular contractions to drive gastrovascular flow may be precluded from establishing lasting symbioses with algae. Indeed, the best-known hydroids with photosynthetic symbionts are the “green hydras.” Since these are not colonial, they do not have sophisticated patterns of muscle-driven gastrovascular flow. While there are colonial hydroids with Symbiodinium symbionts [61], the characteristics of the gastrovascular flow in these colonies remain unknown. Exploring metabolic signaling in such symbiotic colonial hydroids would provide an obvious test of this hypothesis. 6. Conclusions Study of metabolic signaling in cnidarians has a rich history, and there is ample evidence that such an approach can significantly illuminate the biology of these basally branching organisms. In particular, mitochondrial ROS appear to have significant signaling functions in both solitary and colonial polyp stages. In parallel, coral bleaching involves the disruption of metabolic signaling between the host and algal symbionts. Viewing both mitochondrial and algal signaling within the same integrated framework will enhance our understanding of both of these processes. Acknowledgement This work is supported by the NSF (IBN-0090580 and EF0531654). References [1] Lane N. Power, sex, suicide: mitochondria and the meaning of life. Oxford: Oxford University Press; 2005.
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