Influence of swim training on cardiac activity,tissue capillarization,and mitochondrial density in muscle tissue of zebrafish larvae

Larval zebrafish (Danio rerio) of two different age classes ("swim-up" larvae, 9 days old; "free-swimming" larvae, 21 days old) were exposed to either an endurance/continuous training or interval training. Control animals were kept in stagnant water. A comparison of cardiac activity of trained (either endurance or interval) and untrained animals at the end of the training regime revealed no differences in heart rate, end-diastolic and end-systolic ventricular volume, and cardiac output. Training also had no influence on the concentration of erythrocytes in the blood. Thus, at the level of total oxygen transport in the blood, training did not provoke any improvement during the first 32 days of development. Significant changes, however, were observed at the tissue level. In free-swimming larvae [i.e., between 21 and 32 days postfertilization (dpf)] endurance training increased the capillarization of both axial muscle caudal to the anus and the tail fin. In addition, mitochondrial density of red and intermediate muscle fibers increased significantly. In contrast to capillarization, even swim-up larvae, trained between 9 and 15 dpf, were affected. The observed increase in mitochondrial content indicates a high demand for oxygen and energy-rich metabolites for oxidative phosphorylation. In older larvae, this is met by the increase in capillarization that improves the blood supply and with it the required oxygen and metabolite supply of muscle tissue. Both of these adaptational changes result in a reduction of diffusion distances (between capillary and muscle fiber as well as mitochondria) and may contribute to a higher resistance toward oxygen deficiency. Furthermore, this study indicates that plasticity of muscle tissue is already established in early stages of development at both the tissue and cellular levels.     

Temperature-dependent development of cardiac activity in unrestrained larvae of the minnow Phoxinus phoxinus

The minnow (Phoxinus phoxinus) was raised up to the stage of swim bladder inflation at temperatures between 10 degrees C and 25 degrees C, and the time of development significantly decreased at higher temperatures. Accordingly, initiation of cardiac activity was observed at day 2 in 25 degrees C animals and at day 4 in 12.5 degrees C animals. Only a minor increase in body mass was observed during the incubation period, and, at the end of the incubation period, animals raised at 25 degrees C did not have a significantly lower body mass compared with animals raised at 15 degrees C. Metabolic activity, determined as the rate of oxygen consumption of a larva, increased from 3.3 to 19.5 nmol/h during development at 15 degrees C and from 5.6 to 47.6 nmol/h during development at 25 degrees C. Heart rate showed a clear correlation to developmental stage as well as to developmental temperature, but at the onset of cardiac activity, diastolic ventricular volume and also stroke volume were higher at the lower temperatures. Furthermore, stroke volume increased with development, except for the group incubated at 12.5 degrees C, in which stroke volume decreased with development. Initial cardiac output showed no correlation to incubation temperature. Although metabolic activity increased severalfold during development from egg to the stage of swim bladder inflation at 15 degrees C and at 25 degrees C, weight-specific cardiac output increased only by approximately 40% with proceeding development. At 12.5 degrees C, cardiac output remained almost constant until opening of the swim bladder. The data support the notion that oxygen transport is not the major function of the circulatory system at this stage of development. The changes in heart rate with temperature appear to be due to the intrinsic properties of the pacemaker; there was no indication for a regulated response.     

raldh2基因阻抑导致斑马鱼心脏发育畸形的机制

目的通过显微注射吗啡啉修饰的反义寡核苷酸(MO)阻抑视黄醛脱氢酶2(raldh2)基因表达,探讨raldh2基因阻抑对斑马鱼胚胎心脏发育的影响及可能的分子机制。方法根据斑马鱼raldh2基因起始密码区域序列设计合成吗啡啉修饰的反义寡核苷酸,采用显微注射方法阻抑斑马鱼胚胎raldh2基因表达。构建raldh2-EG-FP重组质粒进一步验证MO的特异性和有效性。分析raldh2基因阻抑后对胚胎发育,尤其心脏表型和功能的影响。通过胚胎整体原位杂交,分析心脏相关nppa和tbx20基因表达模式以及raldh2阻抑后对其表达的影响。结果显微注射raldh2-MO能有效地特异地阻抑斑马鱼胚胎raldh2基因表达,raldh2-MO对胚胎发育影响呈剂量依赖性。raldh2基因阻抑可导致胚胎心脏发育畸形,干扰正常的房室分化和向右环化,导致房室瓣血液反流。与野生型胚胎比较,raldh2基因阻抑组胚胎心率和心室收缩分数降低(P<0.05),心功能受损。整体原位杂交结果显示raldh2基因阻抑后nppa基因表达改变,心室部位nppa表达清晰,而心房部位表达减弱。tbx20基因在心脏、运动神经元、顶盖及视网膜表达,raldh2基因阻抑后,tbx20表达下调,在心脏表达减弱,以心房和流出道部位更显著。结论 raldh2基因在心脏早期发育的多个环节发挥重要作用,影响房室分化、心管环化和心肌收缩等。在心脏发育过程中nppa和tbx20基因表达受到raldh2基因调控,可能参与RA信号缺乏导致心脏畸形的潜在分子机制。     

Impact of chronic sub-lethal methylparaben exposure on cardiac hypoxia and alterations in neuroendocrine factors in zebrafish model

Molecular Biology Reports - Endocrine-disrupting chemicals have been shown to cause toxicity in different systems of the body including the endocrine, cardiovascular and nervous systems. This study...     

Ontogeny of intestinal motility in correlation to neuronal development in zebrafish embryos and larvae

For the first time, spontaneous intestinal activity was demonstrated and quantified before the onset of exogenous feeding in zebrafish Danio rerio embryos and larvae in vivo , using digital motion analysis. At 3 days post fertilization (dpf), erratic and spontaneous contraction waves were observed in the gut. Later (4–7 dpf), more distinct contraction patterns were distinguished, and anterograde and retrograde contraction waves projecting anally and orally along the intestine, respectively, as well as local rectal contraction waves could be identified and quantified. The frequency of both anterograde intestinal and local rectal contractions increased significantly during the first days of development. There was a tendency towards shorter anterograde contraction waves in the first dpf stage investigated, but the velocity of the waves did not differ significantly between the different dpf stages. The presence of developing neurones in the gut of zebrafish was established using immunohistochemistry, staining for a suite of marker proteins (Hu C/D, HNK-1 and acetylated tubulin). Structural neurones were present in the developing gut from the first dpf stage investigated (2 dpf). In conclusion, during the period (3–7 dpf) when erratic contraction waves turn into a more organized pattern of motility there is also a pronounced development of the innervation, suggesting a correlation in time of the development of gut motility and its neuronal control.     

Measuring the optokinetic response of zebrafish larvae

Our laboratory screens for visual mutants by examining larval eye movements in response to rotating illuminated stripes. This behavior, which is termed an optokinetic response (OKR), is a reflex that appears in zebrafish at the same time as the development of the visual system. The OKR can be accurately measured by 4 d post-fertilization, which is the age when larvae begin foraging for food. The OKR requires approximately 1 min per larva analyzed. After identifying fish with defective eye movements, we conduct secondary screens (such as histological analysis and electroretinography) to identify the subset of fish with disruptions in the function of the outer retina. This paper describes our protocol for the OKR. Our setup is simple to construct and the materials needed are inexpensive. This makes our system especially useful for new undergraduate and graduate students, as well as introductory science lecturers.     

Influence of age, sex and hypoxia on plasma dopamine-beta-hydroxylase activity in the rat

Using rats (Wistar strain) of our own breed, we studied dopamine-beta-hydroxylase (E.C. 1.14.17.1) (DBH) activity in the plasma of animals of different ages (in correlation to sex) under normal conditions and after exposure to altitude hypoxia (corresponding to 7000 or 9000 m and lasting 20 min). The enzyme was determined by the method of Kato et al. (1974). We found that the given plasma enzyme activity was significantly higher in females than in males, throughout the whole life-span. In addition, we found that minimum activity was reached on about the 14th and 21st day of postnatal life and again on the 40th day, while maximum activity was recorded at the ages of 5, 30 and 35 days and in adult rats. In adult animals (males and females), exposure to altitude hypoxia was followed by a statistically significant increase in plasma DBH activity, which was much more pronounced in females than in males. In males, 240 min after terminating hypoxia plasma DBH activity had returned to normal, but in females it was still significantly raised; after 48 h, plasma DBH activity in females was identical to the activity before exposure to hypoxia. In rats aged 5 and 35 days, hypoxia evoked a fluctuating response. A decrease in activity immediately after terminating hypoxia was followed at 60 min by a return to normal, but at 240 min there was again a significant decrease. In 21-day-old rats, hypoxia did not induce any significant change in plasma DBH activity (the initial activity level in this group was very low).     

Effect of intermittent high altitude hypoxia on the structure and enzymatic activity of cardiac myosin

The time course of structural and enzymatic changes in cardiac myosin was studied in the right and left ventricle of rats exposed to intermittent high altitude (IHA) hypoxia. In the controls, ATPase activity and myosin structure in both ventricles was the same. After the third exposure to simulated high altitude (2 600 m), myosin enzymatic activity rose significantly in the left ventricle and a significant right-left difference appeared. In the next phase of adaptation (11 exposures, 6 000 m), myosin ATPase activity fell in both ventricles and the right-left difference disappeared. After the 16th exposure (7 000 m), enzymatic activity increased again in both ventricles and attained control values. IHA also produced significant structural changes in cardiac myosin, particularly in the rigaht ventricle. The changes were characterized by the formation of myosin aggregates with significantly lower ATPase activity that the myosin monomer. The time course and localization of structural and enzymatic changes in cardiac myosin corresponded to the morphological damage to the heart fibres.     

Ventilatory acclimatization to hypoxia is not dependent on arterial hypoxemia

Goats were prepared so that one carotid body (CB) could be perfused with blood in which the gas tensions could be controlled independently from the blood perfusing the systemic arterial system, including the brain. Since one CB is functionally adequate, the nonperfused CB was excised. To determine whether systemic arterial hypoxemia is necessary for ventilatory acclimatization to hypoxia (VAH), the CB was perfused with hypoxic normocapnic blood for 6 h [means +/- SE: partial pressure of carotid body O2 (PcbO2), 40.6 +/- 0.3 Torr; partial pressure of carotid body CO2 (PcbCO2), 38.8 +/- 0.2 Torr] while the awake goat breathed room air to maintain systemic arterial normoxia. In control periods before and after CB hypoxia the CB was perfused with hyperoxic normocapnic blood. Changes in arterial PCO2 (PaCO2) were used as an index of changes in ventilation. Acute hypoxia (0.5 h of hypoxic perfusion) resulted in hyperventilation sufficient to reduce average PaCO2 by 6.7 Torr from control (P less than 0.05). Over the subsequent 5.5 h of hypoxic perfusion, average PaCO2 decreased further, reaching 4.8 Torr below that observed acutely (P less than 0.05). Acute CB hyperoxic perfusion (20 min) following 6 h of hypoxia resulted in only partial restoration of PaCO2 toward control values; PaCO2 remained 7.9 Torr below control (P less than 0.05). The progressive hyperventilation that occurred during and after 6 h of CB hypoxia with concomitant systemic normoxia is similar to that occurring with total body hypoxia. We conclude that systemic (and probably brain) hypoxia is not a necessary requisite for VAH.     

Factors that render the kidney susceptible to tissue hypoxia in hypoxemia

To better understand what makes the kidney susceptible to tissue hypoxia, we compared, in the rabbit kidney and hindlimb, the ability of feedback mechanisms governing oxygen consumption (Vo(2)) and oxygen delivery (Do(2)) to attenuate tissue hypoxia during hypoxemia. In the kidney (cortex and medulla) and hindlimb (biceps femoris muscle), we determined responses of whole organ blood flow and Vo(2), and local perfusion and tissue Po(2), to reductions in Do(2) mediated by graded systemic hypoxemia. Progressive hypoxemia reduced tissue Po(2) similarly in the renal cortex, renal medulla, and biceps femoris. Falls in tissue Po(2) could be detected when arterial oxygen content was reduced by as little as 4-8%. Vo(2) remained stable during progressive hypoxemia, only tending to fall once arterial oxygen content was reduced by 55% for the kidney or 42% for the hindlimb. Even then, the fall in renal Vo(2) could be accounted for by reduced oxygen demand for sodium transport rather than limited oxygen availability. Hindlimb blood flow and local biceps femoris perfusion increased progressively during graded hypoxia. In contrast, neither total renal blood flow nor cortical or medullary perfusion was altered by hypoxemia. Our data suggest that the absence in the kidney of hyperemic responses to hypoxia, and the insensitivity of renal Vo(2) to limited oxygen availability, contribute to kidney hypoxia during hypoxemia. The susceptibility of the kidney to tissue hypoxia, even in relatively mild hypoxemia, may have important implications for the progression of kidney disease, particularly in patients at high altitude or with chronic obstructive pulmonary disease.     

Suboptimal temperature-dependent changes in the brain development and activity in Galleria mellonella larvae

The development of Galleria mellonella larvae is strongly affected by suboptimal temperature (18°C). One-day-old last-instar larvae react to 18°C with the arrest of further development for several months described as facultative larval diapause. The aim of this study was to find what type of changes, if any, in the brain correlate with the larval diapause induced by suboptimal temperature. Morphological analysis demonstrated the gradual inhibition of brain development. Paraldehyde-fuchsin (PAF) staining revealed cyclicity in the activity of the medial neurosecretory cells (M-NSC) in the larval brain. SDS-PAGE was used to examine the brain proteins of larvae reared at 30°C and at 18°C. The rate of protein synthesis in the brain of the last instar larvae kept at 18°C, measured as l -[³⁵S]methionine incorporation during 2-h incubation in vitro, was only about 40% of the value characteristic for this tissue during normal development (at 30°C). Despite decrease in the rate of total protein synthesis, suboptimal temperature induced an increase in the level of two major brain proteins: 112 and 84 kDa. In SDS-PAGE analysis, these two proteins appear 21–28 days after transfer to the lower temperature. Whether these proteins are specific for induction of larval diapause of Galleria mellonella remains to be further investigated. Arch. Insect Biochem. Physiol. 38:66–73, 1998. © 1998 Wiley-Liss, Inc.     

Skeletal muscle regeneration in Xenopus tadpoles and zebrafish larvae

Background

Mammals are not able to restore lost appendages, while many amphibians are. One important question about epimorphic regeneration is related to the origin of the new tissues and whether they come from mature cells via dedifferentiation and/or from stem cells. Several studies in urodele amphibians (salamanders) indicate that, after limb or tail amputation, the multinucleated muscle fibres do dedifferentiate by fragmentation and proliferation, thereby contributing to the regenerate. In Xenopus laevis tadpoles, however, it was shown that muscle fibres do not contribute directly to the tail regenerate. We set out to study whether dedifferentiation was present during muscle regeneration of the tadpole limb and zebrafish larval tail, mainly by cell tracing and histological observations.

Results

Cell tracing and histological observations indicate that zebrafish tail muscle do not dedifferentiate during regeneration. Technical limitations did not allow us to trace tadpole limb cells, nevertheless we observed no signs of dedifferentiation histologically. However, ultrastructural and gene expression analysis of regenerating muscle in tadpole tail revealed an unexpected dedifferentiation phenotype. Further histological studies showed that dedifferentiating tail fibres did not enter the cell cycle and in vivo cell tracing revealed no evidences of muscle fibre fragmentation. In addition, our results indicate that this incomplete dedifferentiation was initiated by the retraction of muscle fibres.

Conclusions

Our results show that complete skeletal muscle dedifferentiation is less common than expected in lower vertebrates. In addition, the discovery of incomplete dedifferentiation in muscle fibres of the tadpole tail stresses the importance of coupling histological studies with in vivo cell tracing experiments to better understand the regenerative mechanisms.     

Endosulfan affects health variables in adult zebrafish (Danio rerio) and induces alterations in larvae development

Adult zebrafish (Danio rerio) were exposed to 0 (control), 0.16 or 0.48μg/L of the insecticide, endosulfan, for 28days. Haematology, whole body ions, thiobarbituric acid reactive substances (TBARS), Na(+)K(+)-ATPase, organ histology and reproduction were assessed in adults. The resulting offspring were examined for latent effects on development (heart rate and morphometrics). On day 14, adult fish exposed to 0.16μg/L endosulfan showed significantly lower red blood cell counts than those exposed to 0.48μg/L endosulfan; adult fish exposed to 0.16 ug/L also showed elevated TBARS compared to controls. Both concentrations of endosulfan caused a 4.0 fold increase in Na(+)K(+)-ATPase activity compared to controls (ANOVA, p<0.05). On day 14, the livers of fish exposed to endosulfan had fewer, enlarged hepatocytes, with cell diameters greater than the controls (ANOVA, p<0.05). Morphological alterations in the progeny of fish exposed to endosulfan were observed. Heart beat frequency was significantly lower in larvae from exposed adults to 0.16 μg/L compared to the control (ANOVA, p<0.05). These findings show that sublethal exposure to endosulfan causes adverse sublethal effects in adult D. rerio, and effects on the development of their offspring.     

Rotation and asymmetric development of the zebrafish heart requires directed migration of cardiac progenitor cells

We have used high-resolution 4D imaging of cardiac progenitor cells (CPCs) in zebrafish to investigate the earliest left-right asymmetric movements during cardiac morphogenesis. Differential migratory behavior within the heart field was observed, resulting in a rotation of the heart tube. The leftward displacement and rotation of the tube requires hyaluronan synthase 2 expression within the CPCs. Furthermore, by reducing or ectopically activating BMP signaling or by implantation of BMP beads we could demonstrate that BMP signaling, which is asymmetrically activated in the lateral plate mesoderm and regulated by early left-right signals, is required to direct CPC migration and cardiac rotation. Together, these results support a model in which CPCs migrate toward a BMP source during development of the linear heart tube, providing a mechanism by which the left-right axis drives asymmetric development of the vertebrate heart.