Brian Bagatto

A Three-Dimensional Functional Assessment of Heart and Vessel Development in the Larva of the Zebrafish (Danio Rerio)

There has been considerable recent interest in the development of the circulation in the zebrafish. Optical techniques typically used to visualize changes in heart size allow measurement of stroke volume during early vertebrate development, but this approach is complicated in zebrafish larvae because of the heart’s irregular shape and its significant change in morphology during the first 6 d of development. By use of a threedimensional integration of the early zebrafish heart and vessels, we have greatly reduced measurement error of stroke volume and cardiac output and have determined the cross‐sectional growth of major vessels in the developing zebrafish larvae. A dramatic 500%–600% increase in cardiac output (from 10 to 50–60 nL min−1) occurs on days 5 and 6 postfertilization in Danio rerio. Cross‐sectional area of key vessels (dorsal artery, caudal artery, dorsal vein) as well as between‐individual variation significantly decreased over the first 6 d of development. Associated with the decrease in cross‐sectional area is a significant increase in red blood cell velocity on days 5 and 6 postfertilization. Together, the three‐dimensional data of the cardiac and vascular systems have shown that the most profound physiological and developmental changes occur in days 5 and 6, which corresponds with the appearance of the adult form of the heart and the transition from diffusive to convective O2 supply to internal tissues.

Developmental Environment Alters Conditional Aggression in Zebrafish

Abstract The developmental environment influences a wide variety of phenotypic traits in the adults of many vertebrates (i.e., developmental plasticity). In this study, we test to see if developmental environment (EDEV) interacts with the adult behavioral environment (EBEHAV) in determining behavioral phenotypes. We reared Zebrafish (Danio rerio) from eggs in either continuously hypoxic or normoxic conditions. We then tested aggression and avoidance (i.e., hiding) levels of fish from each developmental treatment in both environments. Developmental environment was a significant source of variation in avoidance behavior while the stimulus environment did not influence avoidance. Without a period of acclimation we found that EBEHAV and an EDEV × EBEHAV interaction were both significant sources of variation. However, when the fish were allowed to physiologically acclimate to the environment for 16 h, aggression level was highest for fish tested in the environment in which they developed. In that case the EDEV × EBEHAV interaction was the only significant source of variation. These results demonstrate that a more complete understanding of phenotypic response can be gained by incorporating environmental conditions across multiple time scales.

Maturation of Cardiovascular Control Mechanisms in the Embryonic Emu (Dromiceius Novaehollandiae)

SUMMARY Our understanding of avian embryonic cardiovascular regulation has been based on studies in chickens. The present study was undertaken to determine if the patterns established in chickens are generally applicable to the emu, a ratite bird species. We studied cardiovascular physiology over the interval from 60% to 90% of the emus 50-day incubation period. During this period, embryonic emus exhibit a slight fall in resting heart rate (from 171 beats min-1 to 154 beats min-1) and a doubling of mean arterial pressure (from 1.2 kPa to 2.6 kPa). Exposures to 15% or 10% O2 initially decreased heart rate during the first period of emu incubation studied [60% of incubation (60%I)] but increased heart rate in the 90%I group. Arterial pressure responded to hypoxia with an initial depression (-1.6 kPa) at 60%I and 70%I but showed no response during the later periods of incubation (80%I and 90%I). In addition, tonic stimulation of both cholinergic and adrenergic (α and β) receptors was present on heart rate at 70%I, with the cholinergic and β-adrenergic tone increasing in strength by 90%I. Arterial pressure was dependent on a constant β-adrenergic and constant α-adrenergic tone from 60%I to 90%I. A comparison with embryonic white leghorn chickens over a similar window of incubation revealed that emus and white leghorn chickens both possess an adrenergic tone on heart rate and pressure but that only emus possess a cholinergic tone on heart rate. Collectively, these data indicate that the maturation of cardiovascular control systems differs between white leghorn chickens and emus, inviting investigation of additional avian species to determine other patterns.

Cadherin2 (N-cadherin) plays an essential role in zebrafish cardiovascular development

BackgroundCadherins are cell surface adhesion molecules that play important roles in development of vertebrate tissues and organs. We studied cadherin2 expression in developing zebrafish heart using in situ hybridization and immunocytochemical methods, and we found that cadherin2 was strongly expressed by the myocardium of the embryonic zebrafish. To gain insight into cadherin2 role in the formation and function of the heart, we analyzed cardiac differentiation and performance in a cadherin2 mutant, glass onion (glo).ResultsWe found that the cadherin2 mutant had enlarged pericardial cavity, disorganized atrium and ventricle, and reduced expression of a ventricular specific marker vmhc. Individual myocardiocytes in the glo mutant embryos became round shaped and loosely aggregated. In vivo measurements of cardiac performance revealed that the mutant heart had significantly reduced heart rate, stroke volume and cardiac output compared to control embryos. Formation of the embryonic vascular system in the glo mutants was also affected.ConclusionOur results suggest that cadherin2 plays an essential role in zebrafish cardiovascular development. Although the exact mechanisms remain unknown as to the formation of the enlarged pericardium and reduced peripheral blood flow, it is clear that myocardiocyte differentiation and physiological cardiovascular performance is impaired when cadherin2 function is disrupted.

Cardiovascular system in larval zebrafish responds to developmental hypoxia in a family specific manner

BackgroundGenetic and environmental variation are both known to influence development. Evolution of a developmental response that is optimized to the environment (adaptive plasticity) requires the existence of genetic variation for that developmental response. In complex traits composed of integrated sets of subsidiary traits, the adaptive process may be slowed by the existence of multiple possible integrated responses. This study tests for family (sibship) specific differences in plastic response to hypoxia in an integrated set of cardiovascular traits in zebrafish.ResultsCardiac output, which is the integrated product of several subsidiary traits, varied highly significantly between families, and families differed significantly in the degree and direction of response to developmental oxygen level. The cardiac output response to oxygen environment was entirely family specific with no significant overall trend due to oxygen level. Constituent physiological variables that contribute to cardiac output all showed significant family specific response to hypoxia. Traits that were not directly related to cardiac output, such as arterial and venous diameter, and red blood cell velocities did not respond to hypoxia in a family specific manner.ConclusionZebrafish families vary in their plastic response to hypoxia. Genetic variation in plastic response to hypoxia may therefore provide the basic ingredient for adaptation to a variable environment. Considerable variation in the degree of familial response to hypoxia exists between different cardiovascular traits that may contribute to cardiac output. It is possible that the integration of several subsidiary traits into cardiac output allows the maintenance of genetic variance in cardiac response.

Metabolism, nitrogen excretion, and heat shock proteins in the central mudminnow (Umbra limi), a facultative air-breathing fish living in a variable environment

The central mudminnow (Umbra limi (Kirtland, 1841)) is a continuous, facultative air-breathing freshwater fish found in swamps of central Canada and northeastern USA. The first goal of this field a...

Leptin expression affects metabolic rate in zebrafish embryos (D. rerio)

We used antisense morpholino oligonucleotide technology to knockdown leptin-(A) gene expression in developing zebrafish embryos and measured its effects on metabolic rate and cardiovascular function. Using two indicators of metabolic rate, oxygen consumption was significantly lower in leptin morphants early in development [<48 hours post-fertilization (hpf)], while acid production was significantly lower in morphants later in development (>48 hpf). Oxygen utilization rates in <48 hpf embryos and acid production in 72 hpf embryos could be rescued to that of wildtype embryos by recombinant leptin coinjected with antisense morpholino. Leptin is established to influence metabolic rate in mammals, and these data suggest leptin signaling also influences metabolic rate in fishes.

Bisphenol A alters the cardiovascular response to hypoxia in Danio rerio embryos

The purpose of this study was to determine if the cardiovascular response to hypoxia was altered by the presence of bisphenol A (BPA) in Danio rerio embryos. It was expected that BPA exposure would affect cardiovascular parameters during hypoxia more than normoxia due to an interaction between BPA and the hypoxia-inducible factor (HIF-1α) pathway. We demonstrate that BPA exposure has a minimal effect during normoxia but can severely affect the cardiovascular system during a hypoxic event. Cardiovascular response was measured in vivo using video microscopy and digital motion analysis. RBC density increased 35% in hypoxia alone but decreased 48% with addition of 0.25mg/L BPA. Tissue vascularization (% coverage) was unaffected by hypoxia alone but decreased 37% with addition of 0.25mg/L BPA. The diameter and RBC velocity of arteries were more sensitive than veins to BPA exposure during both normoxia and hypoxia. Arterial RBC velocity decreased 42% during normoxia and 52% during hypoxia with 1mg/L BPA. This decrease in velocity may in part be due to the 86% decrease in heart rate (ƒH) observed during co-exposure to hypoxia and 5mg/L BPA. While stroke volume (SV) was unaffected by treatment, cardiac output (Q) decreased by 69% with co-exposure. ƒH and Q were not affected by BPA exposure during normoxia. Development ultimately slowed by 146% and mortality rates were 95% during hypoxia when exposed to 5mg/L BPA. Our results show for the first time that BPA exposure alters the cardiovascular system during hypoxia more so than normoxia.

Ontogenetic oxygen changes alter zebra fish size, behavior, and blood glucose.

Four male and four female zebra fish were crossed in all possible combinations, resulting in 389 offspring. These offspring were divided among four treatments: normoxia for 90 d, hypoxia for 90 d, normoxia for 30 d followed by hypoxia for 60 d, and hypoxia for 30 d followed by normoxia for 60 d. The effects of early oxygen environment, later oxygen environment, and genotype were then assessed with respect to zebra fish behavior, size, and blood glucose. Fish were tested in an arena where they could shoal with conspecifics before, during, and after the introduction of a novel stimulus. Blood glucose and size were also measured. Early oxygen environment influenced fish size, time spent swimming, and reactivity to a novel stimulus. Environmentally induced plasticity was predominate, with little evidence of among-sire variation for any of the measured parameters.

Physiological variability in yearling alligators: Clutch differences at rest and during activity

The adult phenotype of an organism is the result of its genotype, the environment, and the interaction between the two. Assessing the relative contribution of these factors to the final adult phenotype continues to occupy researchers. Studies have shown clutch effects early in development but few have investigated the persistence of clutch effects on a longer time scale. Five clutches of American alligators were reared for 1 year in a common environment then assessed for the presence of clutch effects as they related to morphological and physiological characteristics. After 1 year, significant clutch effects were evident in all size related variables despite open access to food. Additionally, lung and liver masses remained different between clutches after animal mass was taken into account. Although clutch had no effect on resting heart rate, it significantly contributed to mean arterial pressure. During swimming and exhaustive exercise, the resulting respiratory and metabolic acidoses were strongly dependent on clutch. Therefore, while the environment can have significant influences on the American alligator from hatching to death, the measureable contribution of genetics to the morphology and physiology of the organism remains evident, even after 1 year of common rearing conditions. It behooves researchers to acknowledge and control for clutch effects when designing experiments.