In response to the lack of a transgenic line of zebrafish labeled with heart-specific fluorescence in vivo to serve as a research model, we cloned a 1.6-kb polymerase chain reaction (PCR) -product containing the upstream sequence (؊870 bp), exon 1 (39 bp), intron 1 (682 bp), and exon 2 (69 bp) of the zebrafish cardiac myosin light chain 2 gene, (cmlc2). A germ-line transmitted zebrafish possessing a green fluorescent heart was generated by injecting this PCR product fused with the green fluorescent protein (GFP) gene with ends consisting of inverted terminal repeats of an adeno-associated virus. Green fluorescence was intensively and specifically expressed in the myocardial cells located both around the heart chambers and the atrioventricular canal. Neither the epicardium nor the endocardium showed fluorescent signals. The GFP expression in the transgenic line faithfully recapitulated with the spatial and temporal expression of the endogenous cmlc2. Promoter analysis showed that the fragment consisting of nucleotides from ؊210 to 34 (؊210/34) was sufficient to drive heart-specific expression, with a ؊210/؊73 motif as a basal promoter and a ؊210/؊174 motif as an element involved in suppressing ectopic (nonheart) expression. Interestingly, a germ-line of zebrafish whose GFP appeared ectopically in all muscle types (heart, skeletal, and smooth) was generated by injecting the fragment including a single nucleotide mutation from G to A at ؊119, evidence that A at ؊119 combined with neighboring nucleotides to create a consensus sequence for binding myocyte-specific enhancer factor-2. Developmental Dynamics 228:30 -40, 2003.

BackgroundEpidermal ionocytes play essential roles in the transepithelial transportation of ions, water, and acid-base balance in fish embryos before their branchial counterparts are fully functional. However, the mechanism controlling epidermal ionocyte specification and differentiation remains unknown.Methodology/Principal FindingsIn zebrafish, we demonstrated that Delta-Notch-mediated lateral inhibition plays a vital role in singling out epidermal ionocyte progenitors from epidermal stem cells. The entire epidermal ionocyte domain of genetic mutants and morphants, which failed to transmit the DeltaC-Notch1a/Notch3 signal from sending cells (epidermal ionocytes) to receiving cells (epidermal stem cells), differentiates into epidermal ionocytes. The low Notch activity in epidermal ionocyte progenitors is permissive for activating winged helix/forkhead box transcription factors of foxi3a and foxi3b. Through gain- and loss-of-function assays, we show that the foxi3a-foxi3b regulatory loop functions as a master regulator to mediate a dual role of specifying epidermal ionocyte progenitors as well as of subsequently promoting differentiation of Na+,K+-ATPase-rich cells and H+-ATPase-rich cells in a concentration-dependent manner.Conclusions/SignificanceThis study provides a framework to show the molecular mechanism controlling epidermal ionocyte specification and differentiation in a low vertebrate for the first time. We propose that the positive regulatory loop between foxi3a and foxi3b not only drives early ionocyte differentiation but also prevents the complete blockage of ionocyte differentiation when the master regulator of foxi3 function is unilaterally compromised.

The noteworthy intensification in the development of nanotechnology has led to the development of various types of nanoparticles. The diverse applications of these nanoparticles make them desirable candidate for areas such as drug delivery, coasmetics, medicine, electronics, and contrast agents for magnetic resonance imaging (MRI) and so on. Iron oxide magnetic nanoparticles are a branch of nanoparticles which is specifically being considered as a contrast agent for MRI as well as targeted drug delivery vehicles, angiogenic therapy and chemotherapy as small size gives them advantage to travel intravascular or intracavity actively for drug delivery. Besides the mentioned advantages, the toxicity of the iron oxide magnetic nanoparticles is still less explored. For in vivo applications magnetic nanoparticles should be nontoxic and compatible with the body fluids. These particles tend to degrade in the body hence there is a need to understand the toxicity of the particles as whole and degraded products interacting within the body. Some nanoparticles have demonstrated toxic effects such inflammation, ulceration, and decreases in growth rate, decline in viability and triggering of neurobehavioral alterations in plants and cell lines as well as in animal models. The cause of nanoparticles’ toxicity is attributed to their specific characteristics of great surface to volume ratio, chemical composition, size, and dosage, retention in body, immunogenicity, organ specific toxicity, breakdown and elimination from the body. In the current review paper, we aim to sum up the current knowledge on the toxic effects of different magnetic nanoparticles on cell lines, marine organisms and rodents. We believe that the comprehensive data can provide significant study parameters and recent developments in the field. Thereafter, collecting profound knowledge on the background of the subject matter, will contribute to drive research in this field in a new sustainable direction.

H(+)-ATPase-rich (HR) cells in zebrafish gills/skin were found to carry out Na+ uptake and acid-base regulation through a mechanism similar to that which occurs in mammalian proximal tubular cells. However, the roles of carbonic anhydrases (CAs) in this mechanism in zebrafish HR cells are still unclear. The present study used a functional genomic approach to identify 20 CA isoforms in zebrafish. By screening with whole mount in situ hybridization, only zca2-like a and zca15a were found to be expressed in specific groups of cells in zebrafish gills/skin, and further analyses by triple in situ hybridization and immunocytochemistry demonstrated specific colocalizations of the two zca isoforms in HR cells. Knockdown of zca2-like a caused no change in and knockdown of zca15a caused an increase in H+ activity at the apical surface of HR cells at 24 h postfertilization (hpf). Later, at 96 hpf, both the zca2-like a and zca15a morphants showed decreased H+ activity and increased Na+ uptake, with concomitant upregulation of znhe3b and downregulation of zatp6v1a (H+-ATPase A-subunit) expressions. Acclimation to both acidic and low-Na+ fresh water caused upregulation of zca15a expression but did not change the zca2-like a mRNA level in zebrafish gills. These results provide molecular physiological evidence to support the roles of these two zCA isoforms in Na+ uptake and acid-base regulation mechanisms in zebrafish HR cells.

The measurement of multiple behavior endpoints in zebrafish can provide informative clues within neurobehavioral field. However, multiple behavior evaluations usually require complicated and costly instrumental settings. Here, we reported a versatile setting that applied ten acrylic tanks arranging into five vertical layers and two horizontal columns to perform multiple behavior assays simultaneously, such as the novel tank diving test, mirror-biting test, social interaction, shoaling, and predator escape assay. In total, ten behavioral performance were collected in a single video, and the XY coordination of fish locomotion can be tracked by using open source software of idTracker and ImageJ. We validated our setting by examining zebrafish behavioral changes after exposure to low dose ethanol (EtOH) for 96 h. Fish were observed staying longer time at bottom of the tank, less mirror biting interest, higher freezing time, less fear in predator test, and tight shoaling behaviors which indicated the anxiogenic effect was induced by low dosage exposure of EtOH in zebrafish. In conclusion, the setting in this study provided a simple, versatile and cost-effective way to assess multiple behavioral endpoints in zebrafish with high reliability and reproducibility for the first time.

Stanniocalcin (STC) formerly called hypocalcin or teleocalcin, is a 50-kDa disulfide-linked homodimeric glycoprotein that was originally identified in fish and secreted from the corpuscles of Stannius (CS). One of the main functions of STC-1 is Ca(2+) uptake inhibition; however, the mechanisms remain unknown. In the present study, we provide molecular evidence to elucidate how zebrafish STC-1 regulates Ca(2+) uptake in zebrafish embryos. In a wide variety of tissues including the kidney, brain, gill, muscle, and skin, zstc-1 was expressed. Incubating zebrafish embryos in low-Ca(2+) (0.02 mM) freshwater stimulated whole body Ca(2+) influx and zebrafish epithelial Ca(2+) channel (zECaC) mRNA expression, while downregulated zstc-1 expression. A morpholino microinjection approach was used to knockdown the zSTC-1 protein, and the results showed that the Ca(2+) content, Ca(2+) influx, and zECaC mRNA expression all increased in morphants. These data suggest that zSTC-1 negatively regulates ECaC gene expression to reduce Ca(2+) uptake in zebrafish embryos.

Plastic pollution is a growing global emergency and it could serve as a geological indicator of the Anthropocene era. Microplastics are potentially more hazardous than macroplastics, as the former can permeate biological membranes. The toxicity of microplastic exposure on humans and aquatic organisms has been documented, but the toxicity and behavioral changes of nanoplastics (NPs) in mammals are scarce. In spite of their small size, nanoplastics have an enormous surface area, which bears the potential to bind even bigger amounts of toxic compounds in comparison to microplastics. Here, we used polystyrene nanoplastics (PS-NPs) (diameter size at ~70 nm) to investigate the neurobehavioral alterations, tissue distribution, accumulation, and specific health risk of nanoplastics in adult zebrafish. The results demonstrated that PS-NPs accumulated in gonads, intestine, liver, and brain with a tissue distribution pattern that was greatly dependent on the size and shape of the NPs particle. Importantly, an analysis of multiple behavior endpoints and different biochemical biomarkers evidenced that PS-NPs exposure induced disturbance of lipid and energy metabolism as well as oxidative stress and tissue accumulation. Pronounced behavior alterations in their locomotion activity, aggressiveness, shoal formation, and predator avoidance behavior were exhibited by the high concentration of the PS-NPs group, along with the dysregulated circadian rhythm locomotion activity after its chronic exposure. Moreover, several important neurotransmitter biomarkers for neurotoxicity investigation were significantly altered after one week of PS-NPs exposure and these significant changes may indicate the potential toxicity from PS-NPs exposure. In addition, after ~1-month incubation, the fluorescence spectroscopy results revealed the accumulation and distribution of PS-NPs across zebrafish tissues, especially in gonads, which would possibly further affect fish reproductive function. Overall, our results provided new evidence for the adverse consequences of PS-NPs-induced behavioral dysregulation and changes at the molecular level that eventually reduce the survival fitness of zebrafish in the ecosystem.

Parkinson's disease (PD) is a neurodegenerative disease that is characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra. However, current treatments for PD are mainly palliative. Recently, researchers discovered that neurotoxins can induce Parkinsonian-like symptoms in zebrafish. No study to date has investigated the characteristics of PD, such as neuroinflammation factors, oxidative stress, or ubiquitin dysfunction, in this model. Therefore, the current study was aimed at utilizing commonly used clinical drugs, minocycline, vitamin E, and Sinemet, to test the usefulness of this model. Previous studies had indicated that DA cell loss was greater with 6-hydroxydopamine (6-OHDA) than with other neurotoxins. Thus, we first challenged zebrafish with 6-OHDA immersion and found a significant reduction in zebrafish locomotor activity; we then reversed the locomotor disruptions by treatment with vitamin E, Sinemet, or minocycline. The present study also analyzed the mRNA expression of parkin, pink1, and cd-11b, because the expression of these molecular targets has been shown to result in attenuation in mammalian models of PD. Vitamin E, Sinemet, and minocycline significantly reversed 6-OHDA-induced changes of parkin, pink1, and cd-11b mRNA expression in zebrafish. Moreover, we assessed tyrosine hydroxylase (TH) expression to confirm the therapeutic effects of vitamin E tested on this PD model and established that vitamin E reversed the 6-OHDA-induced damage on TH expression. Our results provide some support for the validity of this in vivo Parkinson's model, and we hope that this model will be more widely used in the future.