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Comparative Biochemistry and Physiology Part A 133 (2003) 547–553 Review Developmental plasticity in the cardiovascular system of fish, with special reference to the zebrafish夞 Bernd Pelster* ¨ Zoologie und Limnologie, Leopold-Franzens-Universitat ¨ Innsbruck, A-6020 Innsbruck, Austria Institut fur Received 2 February 2002; received in revised form 22 May 2002; accepted 28 June 2002 Abstract During development the circulatory system of vertebrates typically starts operating earlier than any other organ. In these early stages, however, blood flow is not yet linked to metabolic requirements of tissues, as is well established for adults. While the autonomic nervous system becomes functional only quite late during development, in the early stages control of blood flow appears to be possible by blood-borne andyor local hormones. This study presents methods based on video-imaging techniques and fluorescence microscopy to visualize cardiac activity, as well as the vascular bed of developing lower vertebrates, and tests the idea that environmental factors, such as hypoxia, may modify cardiac activity, or even the early formation of blood vessels in embryos and larvae. In zebrafish larvae, adaptations of cardiovascular activity to chronic hypoxia become visible shortly after hatching, and the formation of some blood vessels is enhanced under chronic hypoxia. Exposure of early larval stages of zebrafish to a constant water current induces physiological adaptations, resulting in enhanced swimming efficiency and increased tolerance towards hypoxia. Furthermore, application of hormones such as NO can modify cardiac activity as well as peripheral resistance, and they can stimulate blood vessel formation. In consequence, even during early development of fish or amphibian larvae, the performance of cardiac muscle and of skeletal muscle can be modified by environmental influences and peripheral resistance can be adjusted. Even blood vessel formation can be stimulated by hypoxia, for example, or by the presence of specific hormones. Thus, at approximately the time of hatching the physiological performance of vertebrate larvae is already determined by the combined action of environmental influences and of genetic information. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Ontogeny; Circulatory system; Heart; Vascularization; Hypoxia; Hypoxemia; Zebrafish; Xenopus 1. Introduction Form and functioning of the circulatory system of animals has attracted attention for several hundred years. Only within recent years has the 夞 This paper was originally presented at ‘Chobe 2001’; The Second International Conference of Comparative Physiology and Biochemistry in Africa, Chobe National Park, Botswana – August 18–24, 2001. Hosted by the Chobe Safari Lodge and the Mowana Safari Lodge, Kasane; and organised by Natural Events Congress Organizing ([email protected]). *Tel.: q43-512-5076180; fax: q43-512-5072930. E-mail address: [email protected] (B. Pelster). development of new methods and technologies opened up the possibility of including embryos and larvae in this research. During development, the circulatory system of vertebrates typically starts operating earlier than any other organ. Observations of mutants that survive for a couple of days without any heartbeat (Chen and Fishman, 1997), and the fact that zebrafish (Danio rerio) or Xenopus larvae develop nicely for at least 1 or 2 weeks in a CO-containing atmosphere (Pelster and Burggren, 1996; Territo and Altimiras, 1998; Territo and Burggren, 1998) raised questions concerning the main function of the circulatory system at this 1095-6433/03/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 2 . 0 0 1 9 4 - 0 548 B. Pelster / Comparative Biochemistry and Physiology Part A 133 (2003) 547–553 early state of development. Several studies raising larvae at various temperatures revealed that the temperature-induced increase in metabolic rate by far exceeded the temperature-induced increase in cardiac output (Mirkovic and Rombough, 1998; ¨ Schonweger et al., 2000). The obvious explanation for these observations was that in early developmental stages blood flow is not yet linked to metabolic requirements of tissues, as is well established for adults. The linkage between metabolism and cardiac activity represents the main drive for adaptations of the cardiovascular system to changing environmental conditions, for example. This raises the question as to whether the cardiovascular system during early development follows a genetic program and is not capable of a co-ordinated response to environmental changes, or whether there is some flexibility in the design and functioning of the embryonic or larval cardiovascular system. 2. Visualization of cardiac performance and of blood vessels To analyze shape and performance of the developing cardiovascular system, the heart and blood vessels must be visualized, but Doppler methods are not suitable to measure blood flow in blood vessels of capillary size (Burggren and Fritsche, 1995). Thus, video-imaging appears to be the method of choice, but the contrast in a video image of living tissues is rather poor and hardly allows for morphometric analysis of the vascular bed. A significant improvement can be achieved by injecting a dye into the vascular system. Weinstein and co-workers (Weinstein et al., 1995; Isogai et al., 2001) injected fluorescent microspheres into the dorsal and ventral arteries of zebrafish larvae and using confocal microangiography a threedimensional reconstruction of the vascular bed was possible. The result is a remarkable atlas of the vascular anatomy of zebrafish larvae at various developmental stages. A second approach to visualize the vascular bed is a molecular approach. DNA constructs coding for fluorescent protein can be inserted into the genome. If introduced under the control of a tissuespecific promoter, expression is restricted to a single tissue. Tie1 and Tie2 are the genes for tyrosine kinase receptors, which are expressed only in endothelial cells. Thus, a DNA construct was generated with green fluorescent protein (GFP) under the control of the endothelium specific promoter Tie2 (Motoike et al., 2000). If this construct is successfully introduced into Xenopus sperm nuclei and these sperm nuclei are then used for fertilization, transgenic Xenopus develop, which, starting at approximately day 3 of development, express GFP exclusively in endothelial cells. The vasculature of these animals is clearly visible in video images, and similar experiments have also been performed with zebrafish embryos (Walsh and Stainier, 2001). The third method to visualize the vascular bed in transparent larvae was developed in our laboratory, and we named it digital motion analysis (Schwerte and Pelster, 2000). Animals are videotaped using an inverted microscope and the vasculature is visualized by digital analysis of these recordings. If the two fields of a video frame are subtracted, any pixel in which no movement occurred will have the grayscale value zero, while in any pixel in which movement occurred the grayscale value will be different from zero. Thus, any movement that occurred within the 20 ms necessary for the acquisition of one field can be visualized. The length of the shifting vectors generated by this subtraction represents a direct measure for the velocity of a moving particle, i.e. an erythrocyte in the vascular system. By accumulation, the differential images generated from several subsequent video frames, a complete trace of the routes on which erythrocytes moved can be obtained. Thus, a cast of the vascular system, except for those tiny vessels that are not entered by erythrocytes, is obtained. Because the grayscale value of any given pixel or any given group of pixels increases with the number of erythrocytes passing it, this method can also be used to visualize the distribution of blood cells in transparent tissues. Compared to the first two methods, this method is much easier to use because it does not require difficult dye injection and fixation of the animals, and a developmental series can even be recorded from a single animal without the necessity of generating transgenic animals. 3. Stimulation of cardiac activity by lack of oxygen In order to test the possible flexibility of the cardiovascular system during development, animals must be exposed to varied environmental conditions, and one example is to raise animals B. Pelster / Comparative Biochemistry and Physiology Part A 133 (2003) 547–553 under conditions of reduced oxygen availability (Orlando and Pinder, 1995; Fritsche and Burggren, 1996). Soon after fertilization, fish eggs are surprisingly resistant towards a lack of oxygen. Embryos of the Arctic charr (Salvelinus alpinus) exposed to anoxia for 8 h show remarkable metabolic depression and a decrease in heart rate of approximately 90% (Pelster, 1999), and zebrafish embryos survive 24 h of anoxia in a state of suspended animation, in which cardiac activity ceases and mitotic activity of the blastomeres is arrested (Padilla and Roth, 2001). The decrease in heart rate and in cardiac output observed in very early stages of fish and amphibians during periods of oxygen deficiency has been attributed to a direct effect of the lack of oxygen on the metabolism of cardiomyocytes (Orlando and Pinder, 1995; Fritsche and Burggren, 1996; Pelster, 1999). In Xenopus larvae, co-ordinated stimulation of cardiac activity in order to enhance convective oxygen transport during periods of hypoxia was only observed in later developmental stages. While survival during episodes of severe hypoxia or even anoxia is only possible by marked metabolic depression or by entering a state of suspended animation, more or less normal developmental progress may be possible during chronic exposure to mild hypoxia. We raised zebrafish until complete opening of the swimbladder at normoxia (i.e. PO2s20 kPa) and at a PO2 of 10 kPa at a temperature of 25, 28 and 31 8C. Heart rate increased with development in normoxic as well as in hypoxic animals at all three temperatures. At 25 and at 28 8C, heart rate was not different in control and in hypoxic animals until days 4 and 5 after fertilization, respectively. Then heart rate was significantly elevated in hypoxic animals, indicating that in hypoxic animals the pacemaker was stimulated either by hormonal or nervous activity. Compared to heart rate, stroke volume appears to be rather temperature-independent, with a Q10 value close to 1.0 in rainbow trout as well as in zebrafish (Mirkovic and Rombough, 1998; Jacob et al., 2002). A comparison of systolic and enddiastolic ventricular volume in zebrafish raised at 25 8C suggested that an increase in diastolic volume in hypoxic animals caused an increase in stroke volume at 4, 5 and 6 days post-fertilization (dpf), which contributed to the increase in cardiac output observed at this point of development. Similar results were obtained at a temperature of 549 Fig. 1. Changes in cardiac output with development within the first 4 (28 and 31 8C) to 5 days (25 8C) of development in zebrafish larvae raised under normoxia and under hypoxia. 25 8C, ns10; 28 8C, ns16; 31 8C, ns12 (modified after Jacob et al., 2002). 28 8C. In animals raised at 31 8C, however, the hypoxia-induced stimulation of cardiac activity was largely reduced compared to the effects observed at lower temperatures. It could well be that the high intrinsic activity of the ventricle at this temperature significantly reduced the scope for any further enhancement of cardiac activity due to environmental conditions. The influence of hypoxia on cardiac activity of zebrafish larvae is nicely demonstrated by comparing the increase in cardiac output observed between the first day of cardiac activity and the time of complete opening of the swimbladder in normoxic and in hypoxic animals (Fig. 1). In normoxic animals, cardiac output increased in animals raised at 31 8C, but there was only a minor increase in animals raised at 25 8C and no increase at all in animals raised at 28 8C. In hypoxic animals, however, cardiac output significantly increased with development at all three temperatures. Therefore, hypoxia clearly stimulates cardiac activity in zebrafish larvae, and the first response is observed at approximately 1 or maybe even 0.5 days after hatching. Before this time, severe hypoxia will only have a direct effect on cell metabolism, resulting in inhibition of ventricular activity, but it cannot yet elicit a co-ordinated response of the heart. Not only cardiac activity, but also the distribution of blood to various tissues and even the capillarization of tissues can be modified by hypoxia in zebrafish larvae. After 7 days of hypox- 550 B. Pelster / Comparative Biochemistry and Physiology Part A 133 (2003) 547–553 Fig. 2. The influence of chronic hypoxia (7 days, PO2s10 kPa) on gut perfusion and blood vessel formation in larval zebrafish. At 7 dpf all larvae raised at normoxia have a perfused gut, but in only 55% of hypoxic animals is blood flow to the gut observed. The number of hypoxic animals showing the presence of intersegmental anastomosis connecting intersegmental vessels in the tail region and the presence of the caudal vascular tree exceeds the number of normoxic animals showing the presence of these blood vessels (S. Grillitsch, T. Schwerte, B. Pelster, unpublished observation). ia, we observed that in all control animals the gut was perfused with blood. However, only in approximately 50% of the hypoxic animals was blood flow to the gut observed (S. Grillitsch, T. Schwerte, B. Pelster, unpublished observation). On the other hand, blood vessel formation was enhanced in hypoxic animals. Soon after hatching, the intersegmental vessels become interconnected by anastomosing vessels. In approximately 50% of the normoxic animals the anastomosis is present at 7 dpf, but approximately 80% of the hypoxic animals show this pattern. In addition, a caudal vascular tree is present in less than 10% of the normoxic animals, but in approximately 15% of the hypoxic animals (Fig. 2). Thus, hypoxia also modifies organ perfusion and blood vessel formation. Shortage of oxygen supply can occur during periods of reduced oxygen availability, but it can also be induced by hypoxemia, i.e. by a reduction in hemoglobin oxygen-carrying capacity. Hypoxemia can be experimentally induced by raising zebrafish in the presence of CO, blocking the hemoglobin oxygen-binding sites, or of phenylhydrazine, which destroys the hemoglobin. Zebrafish raised until 4 or 5 dpf under these conditions have a similar oxygen consumption compared to control animals (Pelster and Burggren, 1996), and the same is true for Xenopus larvae raised under chronic CO exposure until Nieuwkoop Faber (NF) stage 54 (Territo and Altimiras, 1998; Territo and Burggren, 1998). Measurements of muscle lactate concentration revealed no indication for a stimulation of anaerobic metabolism in zebrafish larvae raised under hypoxemia until 14 dpf (Jacob et al., 2002), and in Xenopus larvae raised under hypoxemia until NF stage 54 (Territo and Burggren, 1998). Cardiac performance of hypoxemic zebrafish is not different from control animals until 2 weeks after fertilization (Pelster and Burggren, 1996; Jacob et al., 2002). Territo and Altimiras (1998) analyzed cardiac performance of Xenopus raised under hypoxic, hypoxemic and hyperoxic conditions and concluded that altering environmental PO2 does not modify global tissue perfusion and cardiac output, but CO exposure enhanced cardiac output in early developmental stages. The obvious conclusion from these considerations is that convective oxygen transport is not necessary in zebrafish larvae until approximately 2 weeks after fertilization if the PO2 gradient through the skin is normal. If this gradient is reduced due to a decrease in environmental PO2, cardiac activity can be stimulated at approximately 0.5 or 1 day after hatching. This appears to be even earlier than in the larger salmonid or Xenopus larvae, where the hypoxic stimulation was not observed before 1 day after hatching or even later. Thus, the time in development at which a coordinated response of the ventricle becomes possible is not only governed by body mass. 4. The influence of chronic swimming activity Hypoxic conditions can also result from increased metabolic activity. This is possible, for example, during periods of exercise, where the increased activity of myosin ATPase significantly stimulates oxygen consumption of the muscle cells. Swim training leads to a modification of growth rate and of food conversion efficiency in adult fish (Wieser et al., 1988; Christiansen and Jobling, 1990). To assess possible influences of chronic swimming activity on metabolism and growth, zebrafish larvae were trained in a swim tunnel system for approximately 15 h every day at a water velocity of 5 bl sy1 (body length per second) (Bagatto et al., 2001). Due to feeding require- B. Pelster / Comparative Biochemistry and Physiology Part A 133 (2003) 547–553 551 (Johnston et al., 1977; De Graaf et al., 1990). Adaptations are also possible on the level of oxygen and nutrient supply to the muscle tissue. This may include mitochondrial density, diffusion distances in muscle tissue and an improvement in convective oxygen transport (Hoppeler and Billeter, 1991). Although thermal tolerance of larvae appears to be lower in larval fish compared to adults and sensitivity towards toxic chemicals appears to be higher than in adult fish (Brett, 1964; Rombough, 1996), early larval stages do have some scope for adaptations to changing environmental conditions. Fig. 3. Active mass-specific oxygen consumption in free swimming zebrafish larvae as a function of water velocity. Larvae were measured after the 11th day of the experimental period of either no training (d) or training at 5 bl sy1 (m) (modified after Bagatto et al., 2001). ments, three different stage groups were trained: yolk sac larvae, which cannot really be trained because they cling to the swim tube and orient towards the current; swim-up fry feeding on Paramecium; and free swimming larvae that feed on Artemia. The training protocol had no influence on growth of the larvae, although yolk absorption was significantly faster in animals exposed to a constant water current. An exciting observation was that, compared to control animals, free swimming larvae after 11 days of training had a higher MO2 at rest, and a significantly lower rate of oxygen consumption in the swim tunnel at any given water velocity (Fig. 3). The higher rate of oxygen consumption at rest may be attributable to an increased activity level at rest. The reduction in MO2 during activity, however, clearly shows that the efficiency of muscular contraction andyor the efficiency of energy conversion have been improved in trained animals. Furthermore, training enhanced the survival of larvae during exposure to low oxygen partial pressures (Bagatto et al., 2001). Exposure of early larval stages of zebrafish to a constant water current therefore induced physiological adaptations, resulting in enhanced swimming efficiency and in increased tolerance towards hypoxia. The nature of these adaptations has not yet been disclosed, but it could, for example, be on the level of enzyme functioning in muscle cells. Myosin ATPase activity or Ca2q pumping might be more efficient, and the activity of enzymes involved in energy metabolism may be involved 5. The influence of hormones Several studies have shown that cholinergic and adrenergic receptors are present and functional in the vascular system of vertebrate larvae at the time of hatching or even earlier (Kimmel, 1992; Pelster et al., 1993; Fritsche, 1997; Fritsche and Jacobsson, 2000; Fritsche et al., 2000; Jacobsson and Fritsche, 1999). In zebrafish larvae at 8 dpf, a redistribution of blood can be achieved, for example by a-adrenergic stimulation (Schwerte and Pelster, 2000). In addition, locally produced hormones such as NO (Fritsche et al., 2000) or endothelin (Schwerte et al., 2001) contribute to the regulation of peripheral resistance in zebrafish and Xenopus larvae. In rainbow trout alevins exposed for up to 4 weeks to the NO donor isosorbide dinitrate (ISDN), significant dilation of the vitelline vein was observed, combined with a decrease in heart rate, but an increase in cardiac output (Eddy et al., 1999). Thus, a redistribution of blood can be achieved in early larval stages by hormonal activity, and the responsiveness of the vasculature to various hormones precedes the functional control of the cardiovascular system via the autonomic nervous system (Fig. 4). Thus, control mechanisms, an essential prerequisite for any adaptational response that may be elicited by environmental perturbations, are available at approximately the time of hatching or even earlier. The vasodilatory effect of NO contributes to changes in local blood flow, and modifications of sheer stress, for example, may in turn contribute to angiogenesis. NO is an important modulator of VEGF-induced vascular permeability and in VEGF-induced angiogenesis (Conway et al., 2001; Fukumura et al., 2001). Chronic exposure of zebrafish to water containing SNP or ISDN, both 552 B. Pelster / Comparative Biochemistry and Physiology Part A 133 (2003) 547–553 Fig. 4. Schematic drawing showing the timetable for the appearance of and or response to: CS, cholinergic stimulation; CT, cholinergic tonus, CNF, cholinergic nerve fibers; AS, adrenergic stimulation; AT, adrenergic tonus; AEC, adrenergic endocrine cells; and ANF, adrenergic nerve fibers, in three species of amphibians (modified after Fritsche and Jacobsson, 2000). substances that release NO into the water, indeed resulted in an earlier appearance of a small blood vessel protruding ventrally from the caudal end of the caudal vein, which was named the caudal vascular tree. In control animals it is first observed at 7 dpf, but only 10% of the animals show this pattern at this time. At 12 dpf, 50% of the animals show this caudal tree. In SNP-incubated animals, this vessel first appears at 5 dpf, and by 7 dpf almost 50% of the animals show it (S. Grillitsch, T. Schwerte, B. Pelster, unpublished observation). These results demonstrate that in early larval stages peripheral resistance, as well as cardiac activity, can be modified by various hormones. NO also appears to exert an effect on blood vessel formation. Although the autonomic nervous system gains control over cardiac activity only late during development (Kimmel, 1992; Protas and Leontieva, 1992; Fritsche, 1997; Jacobsson and Fritsche, 1999), hormonal control is apparently established much earlier. This hormonal control, however, should enable the larvae to respond to environmental changes. 6. Conclusion and adjustments of cardiac activity to changing environmental conditions in adult vertebrates, is not established in embryonic and early larval stages of fish or amphibians. Accordingly, at normoxic environmental PO2, convective oxygen transport in the blood is not required until well after hatching. Nevertheless, hormonal control of peripheral resistance and of cardiac activity is established prior to hatching. Using this control system, larvae are capable of modifying cardiac activity and peripheral resistance in response to environmental changes, such as hypoxia or forced exercise, even at a time when convective oxygen transport is not yet essential to assure oxygen supply to body tissues. Thus, there is some flexibility in the design and functioning of the embryonic or larval cardiovascular system, and it is not operating simply on the basis of a given genetic program. Acknowledgments Parts of the study were financially supported by ¨ the Fonds zur Forderung der wissenschaftlichen Forschung (FWF, P12571-BIO and P14976-BIO). References There is a bulk of evidence demonstrating that the linkage between metabolism and cardiac activity, which represents the main drive for adaptations Bagatto, B., Pelster, B., Burggren, W.W., 2001. Growth and metabolism of larval zebrafish: effects of swim training. J. Exp. Biol. 204, 4335–4343. B. 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