Ontogenetic development of erythropoiesis can be studied non-invasively in GATA-1:DsRed transgenic zebrafish.

For the erythroid cell lineage development in vertebrates, GATA-1 transcription factor is essential. In our report, we have demonstrated that the approximate developmental status of erythrocytes and the progression of blood formation can be studied non-invasively in GATA-1:DsRed transgenic zebrafish (Danio rerio) embryo and larva by characterization of fluorescence luminance spectra. The study was carried out for animals maintained under normoxic and hypoxic (152 and 20 torr PO(2) respectively) conditions up to 10 days post-fertilization (dpf) and total blood cell concentrations and fluorescent cells' percentage were determined for this purpose. The erythroids were classified into five intensity stages (IS) on the basis of their fluorescence intensity. The luminescent cells with medium intensities (IS3) in normoxic animals were found throughout 2 to 10 dpf although in lower quantity while in hypoxic group they appeared from 5 dpf to 10 dpf showing a maximum of 15% of the total luminescent cells at 8 dpf. The total blood cell concentration dropped after 8 dpf in contrast to hypoxic group which showed further increasing trend. The fluorescent cells' percentage in normoxic group was generally higher as compared to the hypoxic ones. Our method successfully defined various stages of erythroid development. An effort was also made to correlate our luminance data (GATA-1 expression) and total blood cell concentrations with Epo mRNA production. Quantitative RT-PCR of 2-15 dpf old zebrafish was carried out for this purpose. Normoxic animals showed 1-3 Epo mRNA copies per ng RNA in contrast to the hypoxic larvae that showed remarkable fluctuation of 1 to 12 Epo mRNA copies per ng RNA during development. The blood volume (aortic diameter) and production time scale proved to be important factors to define the relationship of Epo mRNA with total blood cell concentration and GATA-1 protein expression respectively.

Chronodisruption increases cardiovascular risk in zebrafish via reduced clearance of senescent erythrocytes.

The circadian clock and the hypoxic signaling pathway play critical roles in physiological homeostasis as well as in pathogenesis. The bi-directionality of the interaction between both pathways has been shown on physiological and only recently also on molecular level. But the consequences of a disturbed circadian rhythm for the hypoxic response and the cardiovascular system have never been addressed in any organism. Here we show that the hypoxic response of animals subjected to chronodisruption is reduced by approximately 30%, as reflected by decreased expression levels of hypoxia inducible factor 1 and its down-stream target genes erythropoietin, responsible for the generation of red blood cells (RBC) and vascular endothelial growth factor, which is essential for proper vascularization. Beside malformations of their vascular beds, chronodisrupted animals surprisingly revealed elevated numbers of senescent erythrocytes under normoxic conditions, due to a reduced clearance rate via apoptosis. Over-aged erythrocytes in turn are characterized by decreased oxygen transport capacities and an increased tendency for aggregation, explaining the higher mortality of chronodisrupted animals observed in our study. The present study shows for the first time that chronodisruption strongly interferes with the hypoxic signalling cascade, increasing the cardiovascular risk in zebrafish due to elevated proportions of senescent erythrocytes. The results might shed new light on the etiology of the increased cardiovascular risk observed among shiftworkers.

Development of the sympatho-vagal balance in the cardiovascular system in zebrafish (Danio rerio) characterized by power spectrum and classical signal analysis.

The development of sympatho-vagal control of cardiac activity was analyzed in zebrafish (Danio rerio) larvae from 2 to 15 days post fertilization (d.p.f.) by pharmacological studies as well as by assessing short term heart rate variability. Changes in heart rate in response to cholinergic and adrenergic receptor stimulation or inhibition were investigated using in situ preparations and digital video-microscopic techniques. The data revealed that the heart responded to adrenergic stimulation starting at 4 d.p.f. and to cholinergic stimulation starting at 5 d.p.f. Atropine application resulted in an increase in heart rate beyond 12 d.p.f., while the inhibitory effect of cholinergic stimulation ceased at this time of development. Adrenergic inhibition (propranolol) reduced heart rate for the first time at 5 d.p.f., but the reduction was only very small (3.8%). Between 5 and 12 d.p.f. propranolol application always resulted in a minor reduction in heart rate, but because the effect was so small it was not always significant. Because the presence of an adrenergic or cholinergic tone may influence the stability of heart rate, we analyzed short-term heart rate variability (HRV). The frequency band width of heart rate variability revealed that HRV increased between 4 d.p.f. and 15 d.p.f. From 13 to 15 d.p.f. atropine reduced the frequency band width of HRV, whereas the combination of atropine and propranolol effectively reduced the frequency band width between 11 and 15 d.p.f. Classical power spectrum analysis using electrocardiograms is not possible in tiny zebrafish larvae and juveniles. It was therefore performed using optical methods, recording cardiac movement and cardiotachograms calculated from these measurements. Whereas heart movements contained frequency components characterizing HRV, the cardiotachogram did not show typical frequency spectra as known from other species.

Copper in Helix pomatia (Gastropoda) is regulated by one single cell type: differently responsive metal pools in rhogocytes.

Like all other animal species, terrestrial pulmonate snails require Cu as an essential trace element. On the other hand, elevated amounts of Cu can exert toxic effects on snails. The homeostatic regulation of Cu must therefore be a pivotal goal of terrestrial pulmonates to survive. Upon administration of Cu, snails accumulate the metal nearly equally in most of their organs. Quantitative studies in connection with HPLC and electrospray ionization mass spectrometry reveal that a certain fraction of Cu in snails is bound to a Cu-metallothionein (Cu-MT) isoform that occurs in most organs at constant concentrations, irrespective of whether the animals had been exposed to physiological or elevated amounts of Cu. In situ hybridization demonstrates that at the cellular level, the Cu-binding MT isoform is exclusively expressed in the so-called pore cells (or rhogocytes), which can be found in all major snail organs. The number of pore cells with Cu-MT mRNA reaction products remains unaffected by Cu exposure. Rhogocytes also are major storage sites of Cu in a granular form, the metal quickly entering the snail tissues upon elevated exposure. The number of rhogocytes with granular Cu precipitations strongly increases upon Cu administration via food. Thus, whereas Cu-MT in the rhogocytes represents a stable pool of Cu that apparently serves physiological tasks, the granular Cu precipitations form a second, quickly inducible, and more easily available pool of the metal that serves Cu regulation by responding to superphysiological metal exposure.

Metabolic and ionic responses of trout hepatocytes to anisosmotic exposure.

Trout hepatocytes exposed to hypo- or hyperosmotic conditions respond by swelling and shrinking, respectively, followed by regulatory volume changes that almost, although not completely, restore cell volume. These anisosmotic conditions have a significant impact on metabolic functions. In hyposmotic medium, oxygen consumption (.VO2) and glucose production rates were significantly reduced, whereas lactate accumulation was not significantly affected. By contrast, hyperosmotic conditions did not affect .VO2 and lactate production but caused a sustained reduction in glucose production. Volume changes were also accompanied by alterations in intracellular free calcium ([Ca2+](i)). At the cell population level, hyposmotic exposure evoked a moderate and slowly developing increase in [Ca2+](i), whereas hyperosmolarity caused a pronounced and sustained increase, which peaked at the time of maximum cell shrinkage but clearly exceeded a mere concentration effect due to volume reduction. Responses of individual cells were highly variable in hyposmotic medium, with only 60% showing a clear increase in [Ca2+](i), while in hyperosmotic conditions all cells displayed elevated [Ca2+](i) levels. A decrease in intracellular pH (pHi) observed in hyposmotic medium was insensitive to EIPA, an inhibitor of Na(+)/H(+) exchange, and SITS, an inhibitor of Cl(-)/HCO(3)(-) exchange, but was prevented in Cl(-)-free medium. In hyperosmotic medium, pHi increased. This alkalinization did not occur under conditions of blocked Na(+)/H(+) exchange and was significantly diminished upon inhibition of Cl(-)/HCO(3)(-) exchange, suggesting an important role of these ion transporters in regulatory volume increase of trout hepatocytes.