Long-term imaging of living adult zebrafish.

The zebrafish has become a widely used animal model due, in large part, to its accessibility to and usefulness for high-resolution optical imaging. Although zebrafish research has historically focused mostly on early development, in recent years the fish has increasingly been used to study regeneration, cancer metastasis, behavior and other processes taking place in juvenile and adult animals. However, imaging of live adult zebrafish is extremely challenging, with survival of adult fish limited to a few tens of minutes using standard imaging methods developed for zebrafish embryos and larvae. Here, we describe a new method for imaging intubated adult zebrafish using a specially designed 3D printed chamber for long-term imaging of adult zebrafish on inverted microscope systems. We demonstrate the utility of this new system by nearly day-long observation of neutrophil recruitment to a wound area in living double-transgenic adult casper zebrafish with fluorescently labeled neutrophils and lymphatic vessels, as well as intubating and imaging the same fish repeatedly. We also show that Mexican cavefish can be intubated and imaged in the same way, demonstrating this method can be used for long-term imaging of adult animals from diverse aquatic species.

A protocol for simultaneous Ca2+ and morphology imaging of brain endothelial tip cells in larval zebrafish.

Endothelial tip cells (ETCs) located at growing blood vessels display high morphological dynamics and associated intracellular Ca2+ activities with different spatiotemporal patterns during migration. Examining the Ca2+ activity and morphological dynamics of ETCs will provide an insight for understanding the mechanism of vascular development in organs, including the brain. Here, we describe a method for simultaneous monitoring and relevant analysis of the Ca2+ activity and morphology of growing brain ETCs in larval zebrafish. For complete details on the use and execution of this protocol, please refer to Liu et al. (2020).

Cisplatin alkylating activity in zebrafish causes resistance to chorionic degradation and inhibition of osteogenesis.

Zebrafish have gained popularity as a model organism due to their rapid, external, and transparent development, high fecundity, and gene homology with higher vertebrate models and humans. Specifically, drug discovery has had high success in the implementation of zebrafish in studies for target discovery, efficacy, and toxicity. However, a major limitation of the zebrafish model is a dependence on waterborne exposure in order to maintain high throughput capabilities. Dose delivery can be impeded by a matrix of N-linked glycoproteins and other polypeptides called the chorion. This acelluar barrier is protective of the developing embryo, and thus new approaches for assessment have involved their removal. In these studies, we explored the chorionic interference of a well-characterized alkylating chemotherapeutic, cisplatin, known to accumulate in the chorion of zebrafish and cause delayed hatching. Our results indicated that increased exposure of cisplatin due to dechorionation did not alter morphological endpoints, although retained confinement reduced total body length and yolk utilization. Additionally, inhibition of osteogenesis visualized with Alizarian Red staining, was observable in dechorionated and non-dechorionated treatment groups. The chorions of cisplatin-treated embryos showed resistance to degradation unless treated with a pronase solution. This may be may be due to cisplatin covalently crosslinking which reinforces the structure. As such, the chorion may play an advantageous role in studies to determine alkylating activity of novel compounds. Furthermore, the expression of zebrafish hatching enzyme was not affected by cisplatin exposure. These studies demonstrate that not only was recapitulation of mechanistic activity supported in zebrafish, but highly relevant off-target toxicities observed in higher vertebrates were identified in zebrafish, regardless of chorionation. Experimental design in drug discovery should consider preliminary studies without dechorionation in order to determine dose impediment or off-target adducting.

Activin A affects feeding by promoting the inner diameter and muscle development of the pharynx and oesophagus in zebrafish (Danio rerio) larvae.

Activin A belongs to the superfamily of transforming growth factor-ß and plays an important role in hormone regulation and tissue development. However, few research studies have been conducted on the effect of activin A on feeding organs in fish. In this study, the zebrafish (Danio rerio) larvae were treated with 1 ng ml-1 activin A for 8 days continuously. The haematoxylin and eosin (H&E) staining section results revealed that the transverse inner diameter of the pharynx and oesophagus significantly increased on the third and eighth days after treatment compared with the control group (P < 0.05). On the eighth day, the cross-sectional area of the pharyngeal muscle increased by 8638 µm2 compared to the control group (P < 0.05). The RNA in situ hybridization results also showed that the expression of skeletal muscle-specific genes (myog and myod) was significantly increased in pharyngeal muscle on the eighth day. Furthermore, the qRT-PCR results showed the expression of gh gene was significantly increased on the eighth day (P < 0.05). At the same time, more larvae in activin A group were able to feed larger brine shrimp (Artemia) than in the control group on the eighth day. In conclusion, activin A could affect feeding by promoting the inner diameter and muscle development of the pharynx and oesophagus in zebrafish larvae. This study is the first to report that the development of the pharynx and oesophagus can directly affect food intake in fish larvae, which provides a theoretical basis for the study of food intake of fish at an early stage.

Developmental and cardiac toxicities of propofol in zebrafish larvae.

Propofol, a commonly used anesthetic, is convenient to use, induces quick effect, enables rapid recovery, and is widely accessible given its stable supply. However, its adverse effects are a concern. Reportedly, propofol exhibits a significant inhibitory effect on the respiratory and circulatory systems. Furthermore, intravenous administration of this drug results in hypotension, rapid heart rate, and respiratory failure. Because many pregnant women are administered propofol during childbirth, it may have a significant negative effect on the development of infants. Propofol can cause considerable developmental neurotoxicity and has known activity on the heart. However, the underling mechanisms of these toxicities remain unclear. In the present study, zebrafish embryos were exposed to propofol at different concentrations (0.05, 0.1, 0.5, 1, 5, 10, and 20 µg/ml) to determine its developmental and cardiac toxicities. Propofol exposure decreased the survival rate and hatchability of zebrafish embryos. Additionally, the embryo malformation rate increased in a concentration-dependent manner. Different types of malformations were observed following propofol administration. The proportion of pericardial cysts increased, whereas the heart rate and size decreased with an increase in propofol concentration. The quantitative reverse-transcription polymerase chain reaction revealed that propofol significantly altered the expression of genes related to cardiac development and functions in zebrafish. Collectively, our findings indicate that propofol exposure induces significant developmental and cardiac toxicities in zebrafish.

Physiological status and functional anatomy of zebra fish (Danio rerio) exposed to various levels of Ga3.

Gallium (Ga) is one of the intermetallic elements that has been used in cancer treatment for a long time. However, Ga compounds are increasingly being used to make high-speed semiconductors and photoelectric devices. The current work investigated physiological and pathological changes in zebra fish (Danio rerio) exposed to various Ga3+ levels (0.55, 1.5, and 3.85 mg/L) over a 14-day test period. Decreases in oxygen consumption were significant (p < 0.05) for groups exposed to 3.85 Ga3+ mg/L; this was associated with the fusion of zebra fish gills lamellae. Serum biochemical changes (including aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase) were consistent with observations of damage to organelles within the hepatocytes at higher Ga3+ exposure levels (1.5 and 3.85 mg/L) in zebra fish. We propose <0.55 Ga3+ mg/L as a biologically safe concentration that can be used to establish water quality criteria for this teleost model.

Observation and characterisation of macrophages in zebrafish liver.

Kupffer cells are liver-resident macrophages that play an important role in mediating immune-related functions in mammals and humans. They are well-known for their capacity to phagocytose large amounts of waste complexes, cell debris, microbial particles and even malignant cells. Location, appearance and functional aspects are important features used to identify these characteristic cells of the liver sinusoid. To-date, there is limited information on the occurrence of macrophages in zebrafish liver. Therefore, we aimed to characterise the ultrastructural and functional aspects of liver-associated macrophages in the zebrafish model by taking advantage of the latest advances in zebrafish genetics and multimodal correlative imaging. Herein, we report on the occurrence of macrophages within the zebrafish liver exhibiting conventional ultrastructural features (e.g. presence of pseudopodia, extensive lysosomal apparatus, a phagolysosome and making up ∼3% of the liver volume). Intriguingly, these cells were not located within the sinusoidal vascular bed of hepatic tissue but instead resided between hepatocytes and lacked phagocytic function. While our results demonstrated the presence and structural similarities with liver macrophages from other experimental models, their functional characteristics were distinctly different from Kupffer cells that have been described in rodents and humans. These findings illustrate that the innate immune system of the zebrafish liver has some distinctly different characteristics compared to other animal experimental models. This conclusion underpins our call for future studies in order to have a better understanding of the physiological role of macrophages residing between the parenchymal cells of the zebrafish liver.

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy.

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

RNA-Seq analysis reveals that spring viraemia of carp virus induces a broad spectrum of PIM kinases in zebrafish kidney that promote viral entry.

PIM kinases are a family of serine/threonine protein kinases that potentiate the progression of the cell cycle and inhibit apoptosis. Because of this, they are considered to be proto-oncogenes, and they represent an interesting target for the development of anticancer drugs. In mammals, three PIM kinases exist (PIM-1, PIM-2 and PIM-3), and different inhibitors have been developed to block their activity. In addition to their involvement in cancer, some publications have reported that the PIM kinases have pro-viral activity, and different mechanisms where PIM kinases favour viral infections have been proposed. Zebrafish possess more than 300 Pim kinase members in their genome, and by using RNA-Seq analysis, we found a high number of Pim kinase genes that were significantly induced after infection with spring viraemia of carp virus (SVCV). Moreover, analysis of the miRNAs modulated by this infection revealed that some of them could be involved in the post-transcriptional regulation of Pim kinase abundance. To elucidate the potential role of the 16 overexpressed Pim kinases in the infectivity of SVCV, we used three different pan-PIM kinase inhibitors (SGI-1776, INCB053914 and AZD1208), and different experiments were conducted both in vitro and in vivo. We observed that the PIM kinase inhibitors had a protective effect against SVCV, indicating that, similar to what is observed in mammals, PIM kinases are beneficial for the virus in zebrafish. Moreover, zebrafish Pim kinases seem to facilitate viral entry into the host cells because when ZF4 cells were pre-incubated with the virus and then were treated with the inhibitors, the protective effect of the inhibitors was abrogated. Although more investigation is necessary, these results show that pan-PIM kinase inhibitors could serve as a useful treatment for preventing the spread of viral diseases.

Morphometric signatures of exposure to endocrine disrupting chemicals in zebrafish eleutheroembryos.

Understanding the mode of action of the different pollutants in human and wildlife health is a key step in environmental risk assessment. The aim of this study was to determine signatures that could link morphological phenotypes to the toxicity mechanisms of four Endocrine Disrupting Chemicals (EDCs): bisphenol A (BPA), perfluorooctanesulfonate potassium salt (PFOS), tributyltin chloride (TBT), and 17-ß-estradiol (E2). Zebrafish (Danio rerio) eleutheroembryos were exposed from 2 to 5 dpf to a wide range of BPA, PFOS, TBT and E2 concentrations. At the end of the exposures several morphometric features were assessed. Common and non-specific effects on larvae pigmentation or swim bladder area were observed after exposures to all compounds. BPA specifically induced yolk sac malabsorption syndrome and altered craniofacial parameters, whereas PFOS had specific effects on the notochord formation presenting higher rates of scoliosis and kyphosis. The main effect of E2 was an increase in the body length of the exposed eleutheroembryos. In the case of TBT, main alterations on the morphological traits were related to developmental delays. When integrating all morphometrical parameters, BPA showed the highest rates of malformations in terms of equilethality, followed by PFOS and, distantly, by TBT and E2. In the case of BPA and PFOS, we were able to relate our results with effects on the transcriptome and metabolome, previously reported. We propose that methodized morphometric analyses in zebrafish embryo model can be used as an inexpensive and easy screening tool to predict modes of action of a wide-range number of contaminants.

Canal neuromasts enhance foraging in zebrafish (Danio rerio).

Aquatic animals commonly sense flow using superficial neuromasts (SNs), which are receptors that extend from the body's surface. The lateral line of fishes is unique among these systems because it additionally possesses receptors, the canal neuromasts (CNs), that are recessed within a channel. The lateral line has inspired the development of engineered sensors and concepts in the analysis of flow fields for submersible navigation. The biophysics of CNs are known to be different from the SNs and thereby offer a distinct submodality. However, it is generally unclear whether CNs play a distinct role in behavior. We therefore tested whether CNs enhance foraging in the dark by zebrafish (Danio rerio), a behavior that we elicited with a vibrating rod. We found that juvenile fish, which have only SNs, bite at this rod at about one-third the rate and from as little as one-third the distance of adults for a high-frequency stimulus (50 < f  < 100 Hz). We used novel techniques for manipulating the lateral line in adults to find that CNs offered only a modest benefit at a lower frequency (20 Hz) and that foraging was mediated entirely by cranial neuromasts. Consistent with our behavioral results, biophysical models predicted CNs to be more than an order of magnitude more sensitive than SNs at high frequencies. This enhancement helps to overcome the rapid spatial decay in high-frequency components in the flow around the stimulus. These findings contrast what has been previously established for fishes that are at least ten-times the length of zebrafish, which use trunk CNs to localize prey. Therefore, CNs generally enhance foraging, but in a manner that varies with the size of the fish and its prey. These results have the potential to improve our understanding of flow sensing in aquatic animals and engineered systems.

Chronic exposure to dichloroacetamide induces biochemical and histopathological changes in the gills of zebrafish.

To evaluate the impact of DCAcAm on zebrafish gill, we measure the responses of antioxidant enzyme (superoxide dismutase, SOD), lipid peroxidation (malondialdehyde, MDA), ATPase (Na+ /K+ -ATPase and Ca2+ /Mg2+ -ATP) and histopathological changes of gill in adult zebrafish, after exposed to different concentrations of DCAcAm (0, 1, 10, 100, and 1000 µg L-1 ) for 30 days. Results indicated that DCAcAm first increased and then decreased SOD activity, and DCAcAm also lowered the activities of Na+ /K+ -ATPase and Ca2+ /Mg2+ -ATPase. These results indicated that high affinity of DCAcAm probably be a main factor, which can damage the structures of enzymes, thereby inhibiting the SOD and ATPase activities. Besides, histopathological investigation results also manifested that chronic exposure to DCAcAm can damage the gill tissues, disrupting the normal function of gills. We conclude that chronic exposure to DCAcAm was harmful to organisms, not only influence gill function, but also further cause damage on the gill tissues.

Vitamin D Stimulates Cardiomyocyte Proliferation and Controls Organ Size and Regeneration in Zebrafish.

Attaining proper organ size during development and regeneration hinges on the activity of mitogenic factors. Here, we performed a large-scale chemical screen in embryonic zebrafish to identify cardiomyocyte mitogens. Although commonly considered anti-proliferative, vitamin D analogs like alfacalcidol had rapid, potent mitogenic effects on embryonic and adult cardiomyocytes in vivo. Moreover, pharmacologic or genetic manipulation of vitamin D signaling controlled proliferation in multiple adult cell types and dictated growth rates in embryonic and juvenile zebrafish. Tissue-specific modulation of vitamin D receptor (VDR) signaling had organ-restricted effects, with cardiac VDR activation causing cardiomegaly. Alfacalcidol enhanced the regenerative response of injured zebrafish hearts, whereas VDR blockade inhibited regeneration. Alfacalcidol activated cardiac expression of genes associated with ErbB2 signaling, while ErbB2 inhibition blunted its effects on cell proliferation. Our findings identify vitamin D as mitogenic for cardiomyocytes and other cell types in zebrafish and indicate a mechanism to regulate organ size and regeneration.

Sprouting and anastomosis in the Drosophila trachea and the vertebrate vasculature: Similarities and differences in cell behaviour.

Branching morphogenesis is a fascinating process whereby a simple network of biological tubes increases its complexity by adding new branches to existing ones, generating an enlarged structure of interconnected tubes. Branching morphogenesis has been studied extensively in animals and much has been learned about the regulation of branching at the cellular and molecular level. Here, we discuss studies of the Drosophila trachea and of the vertebrate vasculature, which have revealed how new branches are formed and connect (anastomose), leading to the establishment of complex tubular networks. We briefly describe the cell behaviour underlying tracheal and vascular branching. Although similar at many levels, the branching and anastomosis processes characterized thus far show a number of differences in cell behaviour, resulting in somewhat different tube architectures in these two organs. We describe the similarities and the differences and discuss them in the context of their possible developmental significance. We finish by highlighting some old and new data, which suggest that live imaging of the development of capillary beds in adult animals might reveal yet unexplored endothelial behaviour of endothelial cells.

Toxicity responses of different organs of zebrafish (Danio rerio) to silver nanoparticles with different particle sizes and surface coatings.

Silver nanoparticles (AgNPs) in aquatic ecosystems are toxic to aquatic organisms. In this study, we aimed to investigate the toxicities and molecular mechanisms of AgNPs with different surface coatings (sodium citrate and polyvinylpyrrolidone) and particle sizes (20 nm and 100 nm) in the gills, intestines, and muscles of zebrafish after 96 h of exposure. Our results indicated that the contribution of particle size to AgNP toxicity was greater than that of the surface coating. Citrate-coated AgNPs were more toxic than polyvinylpyrrolidone-coated AgNPs, and 20-nm AgNPs were more toxic than 100-nm AgNPs. The toxic effects of AgNPs to the tissues were in the order intestines > gills > muscles. Differential expression of genes with the different AgNPs confirmed that they had toxic effects in the zebrafish tissues at the molecular level. Our comprehensive comparison of the toxicities of different AgNPs to aquatic ecosystems will be helpful for further risk assessments of AgNPs.

Comparison of efficacy and toxicity of bevacizumab, endostar and apatinib in transgenic and human lung cancer xenograftzebrafish model.

The poor prognosis in non-small-cell lung cancer has driven the development of novel targeted therapies. Vascular endothelial growth factor is the most potent force in mediating tumor angiogenesis, and many angiogenesis inhibitors have been developed for oncology treatment. We performed a study to characterize the efficacy, safety and tumor suppression of three lung cancer related anti-angiogenic drugs (bevacizumab, endostar and apatinib) using transgenic zebrafish embryo and human lung cancer xenotransplantation model. All three drugs demonstrated remarkable angiogenesis and tumor inhibition effect in the zebrafish model, within the nonlethal dose range. Endostar and bevacizumab showed competitive anti-tumor efficacy. The anti-tumor performance of apatinib was hamstrung by its elevated toxicity at 35 °C. The addition of pemetrexed to anti-angiogenesis therapy had no obvious additional benefit in tumors.

Assessment of raw and ozonated oil sands process-affected water exposure in developing zebrafish: Associating morphological changes with gene expression.

With the ever-increasing amounts of oil sands process-affected water (OSPW) accumulating from Canada's oil sands operations, its eventual release must be considered. As OSPW has been found to be both acutely and chronically toxic to aquatic organisms, remediation processes must be developed to lower its toxicity. Ozone treatment is currently being studied as a tool to facilitate the removal of organic constituents associated with toxicity. Biomarkers (e.g. gene expression) are commonly used when studying the effects of environmental contaminants, however, they are not always indicative of adverse effects at the whole organism level. In this study, we assessed the effects of OSPW exposure on developing zebrafish by linking gene expression to relevant cellular and whole organism level endpoints. We also investigated whether or not ozone treatment decreased biomarkers and any associated toxicity observed from OSPW exposure. The concentrations of classical naphthenic acids in the raw and ozonated OSPW used in this study were 16.9 mg/L and 0.6 mg/L, respectively. Ozone treatment reduced the total amount of naphthenic acids (NAs) in the OSPW sample by 92%. We found that exposure to both raw and ozonated OSPW had no effect on the survival of zebrafish embryos. The expression levels of biotransformation genes CYP1A and CYP1B were induced by raw OSPW exposure, with CYP1B being more highly expressed than CYP1A. In contrast, ozonated OSPW exposure did not increase the expression of CYP1A and only slightly induced CYP1B. A decrease in cardiac development and function genes (NKX2.5 and APT2a2a) was not associates with large changes in heart rate, arrhythmia or heart size. We did not find any indications of craniofacial abnormalities or of increased occurrence of apoptotic cells. Overall, our study found that OSPW was not overtly toxic to zebrafish embryos.

Connectomics of the zebrafish's lateral-line neuromast reveals wiring and miswiring in a simple microcircuit.

The lateral-line neuromast of the zebrafish displays a restricted, consistent pattern of innervation that facilitates the comparison of microcircuits across individuals, developmental stages, and genotypes. We used serial blockface scanning electron microscopy to determine from multiple specimens the neuromast connectome, a comprehensive set of connections between hair cells and afferent and efferent nerve fibers. This analysis delineated a complex but consistent wiring pattern with three striking characteristics: each nerve terminal is highly specific in receiving innervation from hair cells of a single directional sensitivity; the innervation is redundant; and the terminals manifest a hierarchy of dominance. Mutation of the canonical planar-cell-polarity gene vangl2, which decouples the asymmetric phenotypes of sibling hair-cell pairs, results in randomly positioned, randomly oriented sibling cells that nonetheless retain specific wiring. Because larvae that overexpress Notch exhibit uniformly oriented, uniformly innervating hair-cell siblings, wiring specificity is mediated by the Notch signaling pathway.