Ontogeny of cardiovascular control in zebrafish (Danio rerio): Effects of developmental environment

The goal of this symposium paper was to identify and quantify developmental plasticity in the onset of cardiovascular responses in the zebrafish. Developmental plasticity was induced by altering the developmental environment in one of three ways: (1) by developing zebrafish in a constant current of 5 body lengths per second, (2) by developing zebrafish at a colder temperature (20 °C), and (3) by developing zebrafish in severe hypoxia (DO = 0.8 mg/L). Early morphological development was significantly affected by each of the treatment environments with hypoxia slowing development the most and producing the highest variation in measurements. Development in constant water current did not significantly affect the timing onset of cardiovascular responses to the pharmacological agents applied. Development at 20 °C significantly delayed the onset of all cardiovascular responses measured by 2–3 days. Development in hypoxia, however, not only delayed onset of all cardiovascular responses, but also shifted the onset relative to the developmental program. Hypoxia clearly has a profound affect on the onset of cardiovascular regulation and it will take many more studies to elucidate the mechanisms by which hypoxia is having its effect. Furthermore, long term studies are also needed to assess whether the plasticity measured in this study is adaptive in the evolutionary sense.     

低氧对斑马鱼胚胎心脏和血管发育及相关基因表达的影响

研究以斑马鱼(Danio rerio)为研究模型,选择心脏和血管荧光标记的2个品系斑马鱼为实验材料,设定低氧和常氧2种水体溶氧条件,用荧光显微镜检测低氧胁迫对胚胎形态结构、心脏和血管外部形态、心率、胚胎躯干部主要血管形成的影响.研究发现低氧导致胚胎存活率低于常氧.低氧不仅滞后胚胎发育,而且造成胚胎形态异常.低氧胁迫后斑...     

Developmental plasticity in the thermal tolerance of zebrafish Danio rerio

To evaluate developmental plasticity in thermal tolerance of zebrafish Danio rerio , common-stock zebrafish were reared from fertilization to adult in the five thermal regimes (two stable, two with constant diel cycles and one stochastic diel cycle) and their thermal tolerance at three acclimation temperatures compared. The energetic cost of developing in the five regimes was assessed by measuring body size over time. While acclimation accounted for most of the variability in thermal tolerance, there were also significant differences among fish reared in the different regimes, regardless of acclimation. Fish reared in more variable environments (as much as ±6° C diel cycle) had a greater tolerance than those from non-variable environments at the same mean temperature. Fish from the more variable environments were also significantly smaller than those from non-variable environments. These results indicate that the thermal history of individual zebrafish induces irreversible changes to the thermal tolerance of adults.     

Behavioral responses of newly hatched zebrafish (Danio rerio) to amino acid chemostimulants

The zebrafish chemosensory systems of olfaction, taste and solitary chemosensory cells (SCCs) are established during the first week after fertilization (a.f.). These systems presumably support the early development of feeding behaviors required as yolk supplies diminish over the same period. Yet there is no previous data reporting early chemosensory responses in zebrafish. We therefore assayed the chemosensory behavior of newly hatched zebrafish on days 3, 4 and 5 a.f. Responses were compared between fish exposed to water alone versus water containing a mixture of 12 amino acids (100 microM each) flowing through a 50 ml test chamber at 4 ml/min; computer-assisted motion analysis was used to quantify responses. Behavioral responses were first observed at day 4 a.f.; the number of fish swimming, their swimming speeds, and their net-to-gross displacement (NGDR) all increased significantly in response to amino acid stimulation. Because taste buds first appear 4-5 days a.f. and the SCCs may not respond to amino acids, these initial chemosensory responses of day 4 fish may be mediated by already established olfactory neurons. The onset of chemosensitivity in day 4 fish corresponded with an easily recognizable developmental phenotype of inactive floating; day 3 fish were inactive and resting on the bottom while day 5 fish were active and moving through the water column. The ease of identifying responsive day 4 fish suggests these animals may be useful for characterizing odorant sensitivity or developmental plasticity or for screening for chemosensory mutations.     

Physiological development of the embryonic and larval crayfish heart

The cardiovascular system is the first system to become functional in a developing animal and must perform key physiological functions even as it develops and grows. The ontogeny of cardiac physiology was studied throughout embryonic and larval developmental stages in the red swamp crayfish Procambarus clarkii using videomicroscopic dimensional analysis. The heart begins to contract by day 13 of development (at 25 degrees C, 20 kPa O(2)). Cardiac output is primarily regulated by changes in heart rate because stroke volume remains relatively constant throughout embryogenesis. Prior to eclosion, heart rate and cardiac output decreased significantly. Previous data suggest that the decrease in cardiac parameters prior to hatching may be due to an oxygen limitation to the embryo. Throughout development, metabolizing mass and embryonic oxygen consumption increased, while egg surface area remained constant. The surface area of the egg membrane is a constraint on gas exchange; this limitation, in combination with the increasing oxygen demand of the embryo, results in an inadequate diffusive supply of oxygen to developing tissues. To determine if the decrease in cardiac function was the result of an internal hypoxia experienced during late embryonic development, early and late-stage embryos were exposed to hyperoxic water (PO(2) = 40 kPa O(2)). Heart rate in late-stage embryos exposed to hyperoxic water increased significantly over control values, which suggests that the suppression in cardiac function observed in late-stage embryos is due to a limited oxygen supply.     

Ventilatory effects of substance P-saporin lesions in the nucleus tractus solitarii of chronically hypoxic rats

During ventilatory acclimatization to hypoxia (VAH), time-dependent increases in ventilation lower Pco(2) levels, and this persists on return to normoxia. We hypothesized that plasticity in the caudal nucleus tractus solitarii (NTS) contributes to VAH, as the NTS receives the first synapse from the carotid body chemoreceptor afferents and also contains CO(2)-sensitive neurons. We lesioned cells in the caudal NTS containing the neurokinin-1 receptor by microinjecting the neurotoxin saporin conjugated to substance P and measured ventilatory responses in awake, unrestrained rats 18 days later. Lesions did not affect hypoxic or hypercapnic ventilatory responses in normoxic control rats, in contrast to published reports for similar lesions in other central chemosensitive areas. Also, lesions did not affect the hypercapnic ventilatory response in chronically hypoxic rats (inspired Po(2) = 90 Torr for 7 days). These results suggest functional differences between central chemoreceptor sites. However, lesions significantly increased ventilation in normoxia or acute hypoxia in chronically hypoxic rats. Hence, chronic hypoxia increases an inhibitory effect of neurokinin-1 receptor neurons in the NTS on ventilatory drive, indicating that these neurons contribute to plasticity during chronic hypoxia, although such plasticity does not explain VAH.     

Effect of temperature on the development of the aquatic stages of Anopheles gambiae sensu stricto (Diptera: Culicidae)

Global warming may affect the future pattern of many arthropod-borne diseases, yet the relationship between temperature and development has been poorly described for many key vectors. Here the development of the aquatic stages of Africa's principal malaria vector, Anopheles gambiae s.s. Giles, is described at different temperatures. Development time from egg to adult was measured under laboratory conditions at constant temperatures between 10 and 40 degrees C. Rate of development from one immature stage to the next increased at higher temperatures to a peak around 28 degrees C and then declined. Adult development rate was greatest between 28 and 32 degrees C, although adult emergence was highest between 22 and 26 degrees C. No adults emerged below 18 degrees C or above 34 degrees C. Non-linear models were used to describe the relationship between developmental rate and temperature, which could be used for developing process-based models of malaria transmission. The utility of these findings is demonstrated by showing that a map where the climate is suitable for the development of aquatic stages of A. gambiae s.s. corresponded closely with the best map of malaria risk currently available for Africa.     

Developmental plasticity in the cardiovascular system of fish,with special reference to the zebrafish

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 and/or 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.     

Cardiovascular regulation during hypoxia in embryos of the domestic chicken Gallus gallus

Renewed interest in the use of the embryonic chicken as a model of perinatal cardiovascular regulation has inspired new questions about the control mechanisms that respond to acute perturbations, such as hypoxia. The objectives of this study were to determine the cardiovascular responses, the regulatory mechanisms involved in those cardiovascular responses, and whether those mechanisms involved the central nervous system (CNS) of embryonic chickens. Heart rate (f(H)) and blood pressure were measured in chicken embryos of different incubation ages during exposure to different levels of hypoxia (15, 10, and 5% O(2)). At all levels of hypoxia and at all developmental ages, a depression of f(H) and arterial pressure was observed, with the exception of day 20 embryos in 15 and 10% O(2). The intensity of the embryonic f(H) and blood pressure responses were directly related to the level of hypoxia used. Muscarinic and alpha-adrenergic receptor stimulation limited the hypoxic hypotension on days 15-19 and 15-21, respectively, as indicated after blockade with atropine and phentolamine. During the final 3 days of incubation, the intensity of the hypoxic hypotension was magnified due to alpha-vasodilation caused by beta-adrenergic and muscarinic receptor stimulation. In 19- to 21-day-old embryos, the f(H) response to hypoxia was limited by alpha-adrenergic receptor stimulation as indicated by the accentuated bradycardia after blockade with phentolamine. Furthermore, on day 21, atropine limited the hypoxic bradycardia, indicating that muscarinic receptors also play a role in the f(H) response at this age. In addition, the muscarinic actions on the heart and the adrenergic effects on the vasculature appeared to occur through a hypoxic-induced direct release from chromaffin tissue and autonomic nerve terminals. Thus, in embryonic chickens, the only cardiovascular response to hypoxia that involves the CNS was the cholinergic regulation of arterial pressure after day 15 of incubation. Therefore, although embryonic chickens and fetal sheep, the standard models of perinatal cardiovascular physiology, respond to hypoxia with a similar redistribution of cardiac output, the underlying mechanisms differ between these species.     

Chronic hypoxic incubation blunts a cardiovascular reflex loop in embryonic American alligator (<Emphasis Type="Italic">Alligator mississippiensis</Emphasis>)

Hypoxia is a naturally occurring environmental challenge for embryonic non-avian reptiles, and this study is the first to investigate the impact of chronic hypoxia on a possible chemoreflex loop in a developing non-avian reptile. We measured heart rate and blood pressure in normoxic and hypoxic-incubated (10% O₂) American alligator embryos (Alligator mississippiensis) at 70 and 90/95% of development. We hypothesized that hypoxic incubation would blunt embryonic alligators’ response to a reflex loop stimulated by phenylbiguanide (PBG), a 5-HT₃ receptor agonist that stimulates vagal pulmonary C-fiber afferents. PBG injection caused a hypotensive bradycardia in 70 and 95% of development embryos (paired t tests, P < 0.05), a response similar to mammals breathing inspired air (all injections made through occlusive catheter in tertiary chorioallantoic membrane artery). Hypoxic incubation blunted the bradycardic response to PBG in embryos at 95% of development (two-way ANOVA, P < 0.01). We also demonstrated that the vagally mediated afferent limb of this reflex can be partially or completely blocked in ovo with a 5-HT₃ receptor blockade using ondansetron hydrochloride dihydrate (OHD), with a ganglionic blockade using hexamethonium, or with a cholinergic blockade using atropine. Atropine eliminated the hypotensive and bradycardic responses to PBG, and OHD and hexamethonium significantly blunted these responses. This cardiovascular reflex mediated by the vagus was affected by hypoxic incubation, suggesting that reptilian sympathetic and parasympathetic reflex loops have the potential for developmental plasticity in response to hypoxia. We suggest that the American alligator, with an extended length of time between each developmental stage relative to avian species, may provide an excellent model to test the cardiorespiratory effects of prolonged exposure to changes in atmospheric gases. This extended period allows for lengthy studies at each stage without the transition to a new stage, and the natural occurrence of hypoxia and hypercapnia in crocodilian nests makes this stress ecologically and evolutionarily relevant.     

A requirement for <Emphasis Type="Italic">kit</Emphasis> in embryonic zebrafish melanocyte differentiation is revealed by melanoblast delay

Exploring differences in gene requirements between species can allow us to delineate basic developmental mechanisms, provide insight into patterns of evolution, and explain heterochronic differences in developmental processes. One example of differences in gene requirements between zebrafish and mammals is the requirement of the kit receptor tyrosine kinase in melanocyte development. kit is required for migration, survival and differentiation of all neural crest-derived melanocytes in mammals. In contrast, zebrafish kit is not required for differentiation of embryonic melanocytes during normal development. When melanoblast development in zebrafish embryos is delayed by injecting morpholinos targeted to the mitfa gene, we show that these delayed melanoblasts fail to differentiate in kit mutants. Thus, we show that there is a kit requirement for melanocyte differentiation in zebrafish when melanoblast development is delayed. Furthermore, we show that kit is not involved in maintaining melanocyte precursors through the developmental delay, but instead is required for differentiation of melanocytes after the block on their development is removed. Finally, we suggest there is a heterochronic shift in the onset of melanocyte differentiation between fish and mouse, and developmental delay of melanoblast development in zebrafish removes this heterochronic difference.Edited by D. Tautz     

Hypoxia-induced developmental plasticity of larval growth,gill and labyrinth organ morphometrics in two anabantoid fish: The facultative air-breather Siamese fighting fish (Betta splendens) and the obligate air-breather the blue gourami (Trichopodus trichopterus)

Larval and juvenile air breathing fish may experience nocturnal and/or seasonal aquatic hypoxia. Yet, whether hypoxia induces respiratory developmental plasticity in larval air breathing fish is uncertain. This study predicted that larvae of two closely related anabantid fish—the facultative air breather the Siamese fighting fish (Betta splendens) and the obligate air breathing blue gourami (Trichopodus trichopterus)—show distinct differences in developmental changes in body, gill, and labyrinth morphology because of their differences in levels of dependency upon air breathing and habitat. Larval populations of both species were reared in normoxia or chronic nocturnal hypoxia from hatching through 35–38 days postfertilization. Gill and labyrinth variables were measured at the onset of air breathing. Betta splendens reared in normoxia possessed larger, more developed gills (~3× greater area) than T. trichopterus at comparable stages. Surface area of the emerging labyrinth, the air breathing organ, was ~ 85% larger in normoxic B. splendens compared to T. trichopterus. Rearing in mild hypoxia stimulated body growth in B. splendens, but neither mild nor severe hypoxia affected growth in T. trichopterus. Condition factor, K (~ 1.3 in B. splendens, 0.7 in T. trichopterus) was unaffected by mild hypoxia in either species, but was reduced by severe hypoxia to <0.9 only in B. splendens. Severe, but not mild, hypoxia decreased branchial surface area in B. splendens by ~40%, but neither hypoxia level affected Trichopodus branchial surface. Mild, but not severe, hypoxia increased labyrinth surface area by 30% in B. splendens. However, as for branchial surface area, labyrinth surface area was not affected in Trichopodus. These differential larval responses to hypoxic rearing suggest that different larval habitats and activity levels are greater factors influencing developmental plasticity than genetic closeness of the two species.     

Long-term effects of the perinatal environment on respiratory control.

The respiratory control system exhibits considerable plasticity, similar to other regions of the nervous system. Plasticity is a persistent change in system behavior triggered by experiences such as changes in neural activity, hypoxia, and/or disease/injury. Although plasticity is observed in animals of all ages, some forms of plasticity appear to be unique to development (i.e., "developmental plasticity"). Developmental plasticity is an alteration in respiratory control induced by experiences during "critical" developmental periods; similar experiences outside the critical period will have little or no lasting effect. Thus complementary experiments on both mature and developing animals are generally needed to verify that the observed plasticity is unique to development. Frequently studied models of developmental plasticity in respiratory control include developmental manipulations of respiratory gas concentrations (O(2) and CO(2)). Environmental factors not specifically associated with breathing may also trigger developmental plasticity, however, including psychological stress or chemicals associated with maternal habits (e.g., nicotine, cocaine). Despite rapid advances in describing models of developmental plasticity in breathing, our understanding of fundamental mechanisms giving rise to such plasticity is poor; mechanistic studies of developmental plasticity are of considerable importance. Developmental plasticity may enable organisms to "fine tune" their phenotype to optimize the performance of this critical homeostatic regulatory system. On the other hand, developmental plasticity could also increase the risk of disease later in life. Future directions for studies concerning the mechanisms and functional implications of developmental plasticity in respiratory motor control are discussed.     

BDE 49 and developmental toxicity in zebrafish

The polybrominated diphenyl ethers (PBDEs) are a group of brominated flame retardants. Human health concerns of these agents have largely centered upon their potential to elicit reproductive and developmental effects. Of the various congeners, BDE 49 (2,2',4,5'-tetrabromodiphenyl ether) has been poorly studied, despite the fact that it is often detected in the tissues of fish and wildlife species. Furthermore, we have previously shown that BDE 49 is a metabolic debromination product of BDE 99 hepatic metabolism in salmon, carp and trout, underscoring the need for a better understanding of biological effects. In the current study, we investigated the developmental toxicity of BDE 49 using the zebrafish (Danio rerio) embryo larval model. Embryo and larval zebrafish were exposed to BDE 49 at either 5 hours post fertilization (hpf) or 24 hpf and monitored for developmental and neurotoxicity. Exposure to BDE 49 at concentrations of 4iμ-32 μM caused a dose-dependent loss in survivorship at 6 days post fertilization (dpf). Morphological impairments were observed prior to the onset of mortality, the most striking of which included severe dorsal curvatures of the tail. The incidence of dorsal tail curvatures was dose and time dependent. Exposure to BDE 49 caused cardiac toxicity as evidenced by a significant reduction in zebrafish heart rates at 6 dpf but not earlier, suggesting that cardiac toxicity was non-specific and associated with physiological stress. Neurobehavioral injury from BDE 49 was evidenced by an impairment of touch-escape responses observed at 5 dpf. Our results indicate that BDE 49 is a developmental toxicant in larval zebrafish that can cause morphological abnormalities and adversely affect neurobehavior. The observed toxicities from BDE 49 were similar in scope to those previously reported for the more common tetrabrominated congener, BDE 47, and also for other lower brominated PBDEs, suggest that these compounds may share similarities in risk to aquatic species.     

Respiratory and cardiovascular responses to acute hypoxia and hyperoxia in internally pipped chicken embryos.

During the first day of hatching, the developing chicken embryo internally pips the air cell and relies on both the lungs and chorioallantoic membrane (CAM) for gas exchange. Our objective in this study was to examine respiratory and cardiovascular responses to acute changes in oxygen at the air cell or the rest of the egg during internal pipping. We measured lung (VO2(lung)) and CAM (VO2(CAM)) oxygen consumption independently before and after 60 min exposure to combinations of hypoxia, hyperoxia, and normoxia to the air cell and the remaining egg. Significant changes in VO2(total) were only observed with combined egg and air cell hypoxia (decreased VO2(total)) or egg hyperoxia and air cell hypoxia (increased VO2(total)). In response to the different O2 treatments, a change in VO2(lung) was compensated by an inverse change in VO2(CAM) of similar magnitude. To test for the underlying mechanism, we focused on ventilation and cardiovascular responses during hypoxic and hyperoxic air cell exposure. Ventilation frequency and minute ventilation (V(E)) were unaffected by changes in air cell O2, but tidal volume (V(T)) increased during hypoxia. Both V(T) and V(E) decreased significantly in response to decreased P(CO2). The right-to-left shunt of blood away from the lungs increased significantly during hypoxic air cell exposure and decreased significantly during hyperoxic exposure. These results demonstrate the internally pipped embryo's ability to control the site of gas exchange by means of altering blood flow between the lungs and CAM.     

Single and multigenerational responses of body mass to atmospheric oxygen concentrations in Drosophila melanogaster : evidence for roles of plasticity and evolution

Greater oxygen availability has been hypothesized to be important in allowing the evolution of larger invertebrates during the Earth’s history, and across aquatic environments. We tested for evolutionary and developmental responses of adult body size of Drosophila melanogaster to hypoxia and hyperoxia. Individually reared flies were smaller in hypoxia, but hyperoxia had no effect. In each of three oxygen treatments (hypoxia, normoxia or hyperoxia) we reared three replicate lines of flies for seven generations, followed by four generations in normoxia. In hypoxia, responses were due primarily to developmental plasticity, as average body size fell in one generation and returned to control values after one to two generations of normoxia. In hyperoxia, flies evolved larger body sizes. Maximal fly mass was reached during the first generation of return from hyperoxia to normoxia. Our results suggest that higher oxygen levels could cause invertebrate species to evolve larger average sizes, rather than simply permitting evolution of giant species.     

NO as a mediator during the early development of the cardiovascular system in the zebrafish

As a general pattern innervation of the cardiovascular system appears late during development in vertebrate embryos, and cardiovascular control may be achieved by hormonal activity in early stages. However, very little is known about the onset of NO-responsiveness during development, which in adult vertebrates is known to play a key function in many physiological processes such as control of vascular tone, neurotransmission, macrophage activity, and angiogenesis. Analysis of the effect of NO on the cardiovascular system in zebrafish (Danio rerio) embryos and larvae revealed almost no effect on cardiac activity during chronic exposure to NO-producing chemicals, whereas vascular reactivity was observed in veins and arteries of the zebrafish in early developmental stages (5-6 days post fertilization). Chronic exposure also modified the development of the vascular system. The presence of an NO donor (sodium nitroprusside) did not change the patterning of the vascular bed, but it induced an earlier appearance of some blood vessels in the trunk region of the zebrafish larvae. The data reveal that NO plays an important role in the development of the cardiovascular system and in the ontogeny of the cardiovascular control system in fish.     

Testing the beneficial acclimation hypothesis and its alternatives for locomotor performance

The beneficial acclimation hypothesis (BAH) is controversial. While physiological work all but assumes that the BAH is true, recent studies have shown that support for the BAH is typically wanting. The latter have been criticized for assessing the benefits of developmental plasticity rather than acclimation. Here we examine the BAH within a strong inference framework for five congeneric species of ameronothroid oribatid mites that occupy marine to terrestrial habitats. We do so by assessing responses of maximum speed, optimum temperature, and performance breadth, measured from -10 degrees C to 35 degrees C, to four treatment temperatures (0 degrees , 5 degrees , 10 degrees , and 15 degrees C). We show that the BAH and its alternatives often make similar empirical predictions. Weak beneficial acclimation is characteristic of one of the more marine species. In the other two upper-shore and marine species, evidence exists for deleterious acclimation and the colder-is-better hypothesis. In the two fully terrestrial species, there is no plasticity. Lack of plasticity is beneficial when cue reliability is low or costs of plasticity are high, and the former seems plausible in terrestrial habitats. However, weak plasticity in the upper-shore/marine species and the absence of plasticity in the terrestrial species might also be a consequence of phylogenetic constraint.